Secreted and transmembrane polypeptides and nucleic acids encoding the same

ABSTRACT

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This application is a continuation of, and claims priority under 35 USC§120 to, U.S. application Ser. No. 10/028,072 filed Dec. 19, 2001 nowabondoned, which is a continuation of, and claims priority under 35 USC§120 to, PCT Application PCT/US00/32678 filed Dec. 1, 2000, which is acontinuation-in-part of, and claims priority under 35 USC §120 to, U.S.application Ser. No. 09/380,138 filed Aug. 25, 1999 now abondoned, whichis the National Stage filed under 35 USC §371 of PCT ApplicationPCT/US99/05028 filed Mar. 8, 1999, which claims priority under 35 USC§119 to U.S. Provisional Application Ser. No. 60/085,697 filed Apr. 15,1998.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides.

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci. 93:7108–7113 (1996); U.S. Pat. No.5,536,637)].

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor or membrane-bound proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a PROpolypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% nucleic acid sequence identity,alternatively at least about 81% nucleic acid sequence identity,alternatively at least about 82% nucleic acid sequence identity,alternatively at least about 83% nucleic acid sequence identity,alternatively at least about 84% nucleic acid sequence identity,alternatively at least about 85% nucleic acid sequence identity,alternatively at least about 86% nucleic acid sequence identity,alternatively at least about 87% nucleic acid sequence identity,alternatively at least about 88% nucleic acid sequence identity,alternatively at least about 89% nucleic acid sequence identity,alternatively at least about 90% nucleic acid sequence identity,alternatively at least about 91% nucleic acid sequence identity,alternatively at least about 92% nucleic acid sequence identity,alternatively at least about 93% nucleic acid sequence identity,alternatively at least about 94% nucleic acid sequence identity,alternatively at least about 95% nucleic acid sequence identity,alternatively at least about 96% nucleic acid sequence identity,alternatively at least about 97% nucleic acid sequence identity,alternatively at least about 98% nucleic acid sequence identity andalternatively at least about 99% nucleic acid sequence identity to (a) aDNA molecule encoding a PRO polypeptide having a full-length amino acidsequence as disclosed herein, an amino acid sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a transmembraneprotein, with or without the signal peptide, as disclosed herein or anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein, or (b) the complement of the DNA moleculeof (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule comprising the coding sequence of afull-length PRO polypeptide cDNA as disclosed herein, the codingsequence of a PRO polypeptide lacking the signal peptide as disclosedherein, the coding sequence of an extracellular domain of atransmembrane PRO polypeptide, with or without the signal peptide, asdisclosed herein or the coding sequence of any other specificallydefined fragment of the full-length amino acid sequence as disclosedherein, or (b) the complement of the DNA molecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) a DNA molecule that encodes the same maturepolypeptide encoded by any of the human protein cDNAs deposited with theATCC as disclosed herein, or (b) the complement of the DNA molecule of(a).

Another aspect the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a PRO polypeptide which iseither transmembrane domain-deleted or transmembrane domain-inactivated,or is complementary to such encoding nucleotide sequence, wherein thetransmembrane domain(s) of such polypeptide are disclosed herein.Therefore, soluble extracellular domains of the herein described PROpolypeptides are contemplated.

Another embodiment is directed to fragments of a PRO polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes, for encoding fragments of a PRO polypeptide thatmay optionally encode a polypeptide comprising a binding site for ananti-PRO antibody or as antisense oligonucleotide probes. Such nucleicacid fragments are usually at least about 10 nucleotides in length,alternatively at least about 15 nucleotides in length, alternatively atleast about 20 nucleotides in length, alternatively at least about 30nucleotides in length, alternatively at least about 40 nucleotides inlength, alternatively at least about 50 nucleotides in length,alternatively at least about 60 nucleotides in length, alternatively atleast about 70 nucleotides in length, alternatively at least about 80nucleotides in length, alternatively at least about 90 nucleotides inlength, alternatively at least about 100 nucleotides in length,alternatively at least about 110 nucleotides in length, alternatively atleast about 120 nucleotides in length, alternatively at least about 130nucleotides in length, alternatively at least about 140 nucleotides inlength, alternatively at least about 150 nucleotides in length,alternatively at least about 160 nucleotides in length, alternatively atleast about 170 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 190 nucleotides inlength, alternatively at least about 200 nucleotides in length,alternatively at least about 250 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 350nucleotides in length, alternatively at least about 400 nucleotides inlength, alternatively at least about 450 nucleotides in length,alternatively at least about 500 nucleotides in length, alternatively atleast about 600 nucleotides in length, alternatively at least about 700nucleotides in length, alternatively at least about 800 nucleotides inlength, alternatively at least about 900 nucleotides in length andalternatively at least about 1000 nucleotides in length, wherein in thiscontext the term “about” means the referenced nucleotide sequence lengthplus or minus 10% of that referenced length. It is noted that novelfragments of a PRO polypeptide-encoding nucleotide sequence may bedetermined in a routine manner by aligning the PRO polypeptide-encodingnucleotide sequence with other known nucleotide sequences using any of anumber of well known sequence alignment programs and determining whichPRO polypeptide-encoding nucleotide sequence fragment(s) are novel. Allof such PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

In another embodiment, the invention provides isolated PRO polypeptideencoded by any of the isolated nucleic acid sequences hereinaboveidentified.

In a certain aspect, the invention concerns an isolated PRO polypeptide,comprising an amino acid sequence having at least about 80% amino acidsequence identity, alternatively at least about 81% amino acid sequenceidentity, alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80% amino acidsequence identity, alternatively at least about 81% amino acid sequenceidentity, alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anamino acid sequence encoded by any of the human protein cDNAs depositedwith the ATCC as disclosed herein.

In a specific aspect, the invention provides an isolated PRO polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand is encoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO polypeptide and recovering the PRO polypeptide from the cellculture.

Another aspect the invention provides an isolated PRO polypeptide whichis either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO polypeptide which comprise contactingthe PRO polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO polypeptide, or an agonist or antagonist of aPRO polypeptide as herein described, or an anti-PRO antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO polypeptide, or an agonist or antagonist thereof as hereinbeforedescribed, or an anti-PRO antibody, for the preparation of a medicamentuseful in the treatment of a condition which is responsive to the PROpolypeptide, an agonist or antagonist thereof or an anti-PRO antibody.

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody which binds,preferably specifically, to any of the above or below describedpolypeptides. Optionally, the antibody is a monoclonal antibody,humanized antibody, antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probeswhich may be useful for isolating genomic and cDNA nucleotide sequences,measuring or detecting expression of an associated gene or as antisenseprobes, wherein those probes may be derived from any of the above orbelow described nucleotide sequences. Preferred probe lengths aredescribed above.

In yet other embodiments, the present invention is directed to methodsof using the PRO polypeptides of the present invention for a variety ofuses based upon the functional biological assay data presented in theExamples below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO177 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA16438-1387”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequencePRO3574 cDNA, wherein SEQ ID NO:3 is a clone designated herein as“DNA19360-2552”.

FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived from thecoding sequence of SEQ ID NO:3 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a native sequencePRO1280 cDNA, wherein SEQ ID NO:5 is a clone designated herein as“DNA33455-1548”.

FIG. 6 shows the amino acid sequence (SEQ ID NO:6) derived from thecoding sequence of SEQ ID NO:5 shown in FIG. 5.

FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a native sequencePRO4984 cDNA, wherein SEQ ID NO:7 is a clone designated herein as“DNA37155-2651”.

FIG. 8 shows the amino acid sequence (SEQ ID NO:8) derived from thecoding sequence of SEQ ID NO:7 shown in FIG. 7.

FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a native sequencePRO4988 cDNA, wherein SEQ ID NO:9 is a clone designated herein as“DNA38269-2654”.

FIG. 10 shows the amino acid sequence (SEQ ID NO:10) derived from thecoding sequence of SEQ ID NO:9 shown in FIG. 9.

FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a native sequencePRO305 cDNA, wherein SEQ ID NO:11 is a clone designated herein as“DNA40619-1220”.

FIG. 12 shows the amino acid sequence (SEQ ID NO:12) derived from thecoding sequence of SEQ ID NO:11 shown in FIG. 11.

FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a native sequencePRO1866 cDNA, wherein SEQ ID NO:13 is a clone designated herein as“DNA44174-2513”.

FIG. 14 shows the amino acid sequence (SEQ ID NO:14) derived from thecoding sequence of SEQ ID NO:13 shown in FIG. 13.

FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a native sequencePRO4996 cDNA, wherein SEQ ID NO:15 is a clone designated herein as“DNA44675-2662”.

FIG. 16 shows the amino acid sequence (SEQ ID NO:16) derived from thecoding sequence of SEQ ID NO:15 shown in FIG. 15.

FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a native sequencePRO4406 cDNA, wherein SEQ ID NO:17 is a clone designated herein as“DNA45408-2615”.

FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived from thecoding sequence of SEQ ID NO:17 shown in FIG. 17.

FIG. 19 shows a nucleotide sequence (SEQ ID NO:19) of a native sequencePRO1120 cDNA, wherein SEQ ID NO:19 is a clone designated herein as“DNA48606-1479”.

FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived from thecoding sequence of SEQ ID NO:19 shown in FIG. 19.

FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a native sequencePRO4990 cDNA, wherein SEQ ID NO:21 is a clone designated herein as“DNA52753-2656”.

FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived from thecoding sequence of SEQ ID NO:21 shown in FIG. 21.

FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a native sequencePRO738 cDNA, wherein SEQ ID NO:23 is a clone designated herein as“DNA53915-1258”.

FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived from thecoding sequence of SEQ ID NO:23 shown in FIG. 23.

FIG. 25 shows a nucleotide sequence (SEQ ID NO:25) of a native sequencePRO3577 cDNA, wherein SEQ ID NO:25 is a clone designated herein as“DNA53991-2553”.

FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived from thecoding sequence of SEQ ID NO:25 shown in FIG. 25.

FIG. 27 shows a nucleotide sequence (SEQ ID NO:27) of a native sequencePRO1879 cDNA, wherein SEQ ID NO:27 is a clone designated herein as“DNA54009-2517”.

FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived from thecoding sequence of SEQ ID NO:27 shown in FIG. 27.

FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a native sequencePRO1471 cDNA, wherein SEQ ID NO:29 is a clone designated herein as“DNA56055-1643”.

FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived from thecoding sequence of SEQ ID NO:29 shown in FIG. 29.

FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a native sequencePRO1114 cDNA, wherein SEQ ID NO:31 is a clone designated herein as“DNA57033-1403”.

FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived from thecoding sequence of SEQ ID NO:31 shown in FIG. 31.

FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a native sequencePRO1076 cDNA, wherein SEQ ID NO:33 is a clone designated herein as“DNA57252-1453”.

FIG. 34 shows the amino acid sequence (SEQ ID NO:34) derived from thecoding sequence of SEQ ID NO:33 shown in FIG. 33.

FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a native sequencePRO1483 cDNA, wherein SEQ ID NO:35 is a clone designated herein as“DNA58799-1652”.

FIG. 36 shows the amino acid sequence (SEQ ID NO:36) derived from thecoding sequence of SEQ ID NO:35 shown in FIG. 35.

FIG. 37 shows a nucleotide sequence (SEQ ID NO:37) of a native sequencePRO4985 cDNA, wherein SEQ ID NO:37 is a clone designated herein as“DNA59770-2652”.

FIG. 38 shows the amino acid sequence (SEQ ID NO:38) derived from thecoding sequence of SEQ ID NO:37 shown in FIG. 37.

FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a native sequencePRO5000 cDNA, wherein SEQ ID NO:39 is a clone designated herein as“DNA59774-2665”.

FIG. 40 shows the amino acid sequence (SEQ ID NO:40) derived from thecoding sequence of SEQ ID NO:39 shown in FIG. 39.

FIG. 41 shows a nucleotide sequence (SEQ ID NO:41) of a native sequencePRO1881 cDNA, wherein SEQ ID NO:41 is a clone designated herein as“DNA60281-2518”.

FIG. 42 shows the amino acid sequence (SEQ ID NO:42) derived from thecoding sequence of SEQ ID NO:41 shown in FIG. 41.

FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a native sequencePRO4314 cDNA, wherein SEQ ID NO:43 is a clone designated herein as“DNA60736-2559”.

FIG. 44 shows the amino acid sequence (SEQ ID NO:44) derived from thecoding sequence of SEQ ID NO:43 shown in FIG. 43.

FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a native sequencePRO4987 cDNA, wherein SEQ ID NO:45 is a clone designated herein as“DNA61875-2653”.

FIG. 46 shows the amino acid sequence (SEQ ID NO:46) derived from thecoding sequence of SEQ ID NO:45 shown in FIG. 45.

FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a native sequencePRO4313 cDNA, wherein SEQ ID NO:47 is a clone designated herein as“DNA62312-2558”.

FIG. 48 shows the amino acid sequence (SEQ ID NO:48) derived from thecoding sequence of SEQ ID NO:47 shown in FIG. 47.

FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a native sequencePRO4799 cDNA, wherein SEQ ID NO:49 is a clone designated herein as“DNA62849-1604”.

FIG. 50 shows the amino acid sequence (SEQ ID NO:50) derived from thecoding sequence of SEQ ID NO:49 shown in FIG. 49.

FIG. 51 shows a nucleotide sequence (SEQ ID NO:51) of a native sequencePRO4995 cDNA, wherein SEQ ID NO:51 is a clone designated herein as“DNA66307-2661”.

FIG. 52 shows the amino acid sequence (SEQ ID NO:52) derived from thecoding sequence of SEQ ID NO:51 shown in FIG. 51.

FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a native sequencePRO1341 cDNA, wherein SEQ ID NO:53 is a clone designated herein as“DNA66677-2535”.

FIG. 54 shows the amino acid sequence (SEQ ID NO:54) derived from thecoding sequence of SEQ ID NO:53 shown in FIG. 53.

FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a native sequencePRO1777 cDNA, wherein SEQ ID NO:55 is a clone designated herein as“DNA71235-1706”.

FIG. 56 shows the amino acid sequence (SEQ ID NO:56) derived from thecoding sequence of SEQ ID NO:55 shown in FIG. 55.

FIG. 57 shows a nucleotide sequence (SEQ ID NO:57) of a native sequencePRO3580 cDNA, wherein SEQ ID NO:57 is a clone designated herein as“DNA71289-2547”.

FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived from thecoding sequence of SEQ ID NO:57 shown in FIG. 57.

FIG. 59 shows a nucleotide sequence (SEQ ID NO:59) of a native sequencePRO1779 cDNA, wherein SEQ ID NO:59 is a clone designated herein as“DNA73775-1707”.

FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived from thecoding sequence of SEQ ID NO:59 shown in FIG. 59.

FIG. 61 shows a nucleotide sequence (SEQ ID NO:61) of a native sequencePRO1754 cDNA, wherein SEQ ID NO:61 is a clone designated herein as“DNA76385-1692”.

FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived from thecoding sequence of SEQ ID NO:61 shown in FIG. 61.

FIG. 63 shows a nucleotide sequence (SEQ ID NO:63) of a native sequencePRO1906 cDNA, wherein SEQ ID NO:63 is a clone designated herein as“DNA76395-2527”.

FIG. 64 shows the amino acid sequence (SEQ ID NO:64) derived from thecoding sequence of SEQ ID NO:63 shown in FIG. 63.

FIG. 65 shows a nucleotide sequence (SEQ ID NO:65) of a native sequencePRO1870 cDNA, wherein SEQ ID NO:65 is a clone designated herein as“DNA77622-2516”.

FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived from thecoding sequence of SEQ ID NO:65 shown in FIG. 65.

FIG. 67 shows a nucleotide sequence (SEQ ID NO:67) of a native sequencePRO4329 cDNA, wherein SEQ ID NO:67 is a clone designated herein as“DNA77629-2573”.

FIG. 68 shows the amino acid sequence (SEQ ID NO:68) derived from thecoding sequence of SEQ ID NO:67 shown in FIG. 67.

FIG. 69 shows a nucleotide sequence (SEQ ID NO:69) of a native sequencePRO4979 cDNA, wherein SEQ ID NO:69 is a clone designated herein as“DNA77645-2648”.

FIG. 70 shows the amino acid sequence (SEQ ID NO:70) derived from thecoding sequence of SEQ ID NO:69 shown in FIG. 69.

FIG. 71 shows a nucleotide sequence (SEQ ID NO:71) of a native sequencePRO1885 cDNA, wherein SEQ ID NO:71 is a clone designated herein as“DNA79302-2521”.

FIG. 72 shows the amino acid sequence (SEQ ID NO:72) derived from thecoding sequence of SEQ ID NO:71 shown in FIG. 71.

FIG. 73 shows a nucleotide sequence (SEQ ID NO:73) of a native sequencePRO1882 cDNA, wherein SEQ ID NO:73 is a clone designated herein as“DNA79865-2519”.

FIG. 74 shows the amino acid sequence (SEQ ID NO:74) derived from thecoding sequence of SEQ ID NO:73 shown in FIG. 73.

FIG. 75 shows a nucleotide sequence (SEQ ID NO:75) of a native sequencePRO4989 cDNA, wherein SEQ ID NO:75 is a clone designated herein as“DNA80135-2655”.

FIG. 76 shows the amino acid sequence (SEQ ID NO:76) derived from thecoding sequence of SEQ ID NO:75 shown in FIG. 75.

FIG. 77 shows a nucleotide sequence (SEQ ID NO:77) of a native sequencePRO4323 cDNA, wherein SEQ ID NO:77 is a clone designated herein as“DNA80794-2568”.

FIG. 78 shows the amino acid sequence (SEQ ID NO:78) derived from thecoding sequence of SEQ ID NO:77 shown in FIG. 77.

FIG. 79 shows a nucleotide sequence (SEQ ID NO:79) of a native sequencePRO1886 cDNA, wherein SEQ ID NO:79 is a clone designated herein as“DNA80796-2523”.

FIG. 80 shows the amino acid sequence (SEQ ID NO:80) derived from thecoding sequence of SEQ ID NO:79 shown in FIG. 79.

FIG. 81 shows a nucleotide sequence (SEQ ID NO:81) of a native sequencePRO4395 cDNA, wherein SEQ ID NO:81 is a clone designated herein as“DNA80840-2605”.

FIG. 82 shows the amino acid sequence (SEQ ID NO:82) derived from thecoding sequence of SEQ ID NO:81 shown in FIG. 81.

FIG. 83 shows a nucleotide sequence (SEQ ID NO:83) of a native sequencePRO1782 cDNA, wherein SEQ ID NO:83 is a clone designated herein as“DNA80899-2501”.

FIG. 84 shows the amino acid sequence (SEQ ID NO:84) derived from thecoding sequence of SEQ ID NO:83 shown in FIG. 83.

FIG. 85 shows a nucleotide sequence (SEQ ID NO:85) of a native sequencePRO4338 cDNA, wherein SEQ ID NO:85 is a clone designated herein as“DNA81228-2580”.

FIG. 86 shows the amino acid sequence (SEQ ID NO:86) derived from thecoding sequence of SEQ ID NO:85 shown in FIG. 85.

FIG. 87 shows a nucleotide sequence (SEQ ID NO:87) of a native sequencePRO4341 cDNA, wherein SEQ ID NO:87 is a clone designated herein as“DNA81761-2583”.

FIG. 88 shows the amino acid sequence (SEQ ID NO:88) derived from thecoding sequence of SEQ ID NO:87 shown in FIG. 87.

FIG. 89 shows a nucleotide sequence (SEQ ID NO:89) of a native sequencePRO5990 cDNA, wherein SEQ ID NO:89 is a clone designated herein as“DNA96042-2682”.

FIG. 90 shows the amino acid sequence (SEQ ID NO:90) derived from thecoding sequence of SEQ ID NO:89 shown in FIG. 89.

FIG. 91 shows a nucleotide sequence (SEQ ID NO:91) of a native sequencePRO3438 cDNA, wherein SEQ ID NO:91 is a clone designated herein as“DNA82364-2538”.

FIG. 92 shows the amino acid sequence (SEQ ID NO:92) derived from thecoding sequence of SEQ ID NO:91 shown in FIG. 91.

FIG. 93 shows a nucleotide sequence (SEQ ID NO:93) of a native sequencePRO4321 cDNA, wherein SEQ ID NO:93 is a clone designated herein as“DNA82424-2566”.

FIG. 94 shows the amino acid sequence (SEQ ID NO:94) derived from thecoding sequence of SEQ ID NO:93 shown in FIG. 93.

FIG. 95 shows a nucleotide sequence (SEQ ID NO:95) of a native sequencePRO4304 cDNA, wherein SEQ ID NO:95 is a clone designated herein as“DNA82430-2557”.

FIG. 96 shows the amino acid sequence (SEQ ID NO:96) derived from thecoding sequence of SEQ ID NO:95 shown in FIG. 95.

FIG. 97 shows a nucleotide sequence (SEQ ID NO:97) of a native sequencePRO1801 cDNA, wherein SEQ ID NO:97 is a clone designated herein as“DNA83500-2506”.

FIG. 98 shows the amino acid sequence (SEQ ID NO:98) derived from thecoding sequence of SEQ ID NO:97 shown in FIG. 97.

FIG. 99 shows a nucleotide sequence (SEQ ID NO:99) of a native sequencePRO4403 cDNA, wherein SEQ ID NO:99 is a clone designated herein as“DNA83509-2612”.

FIG. 100 shows the amino acid sequence (SEQ ID NO:100) derived from thecoding sequence of SEQ ID NO:99 shown in FIG. 99.

FIG. 101 shows a nucleotide sequence (SEQ ID NO:101) of a nativesequence PRO4324 cDNA, wherein SEQ ID NO:101 is a clone designatedherein as “DNA83560-2569”.

FIG. 102 shows the amino acid sequence (SEQ ID NO:102) derived from thecoding sequence of SEQ ID NO:101 shown in FIG. 101.

FIG. 103 shows a nucleotide sequence (SEQ ID NO:103) of a nativesequence PRO4303 cDNA, wherein SEQ ID NO:103 is a clone designatedherein as “DNA84139-2555”.

FIG. 104 shows the amino acid sequence (SEQ ID NO:104) derived from thecoding sequence of SEQ ID NO:103 shown in FIG. 103.

FIG. 105 shows a nucleotide sequence (SEQ ID NO:105) of a nativesequence PRO4305 cDNA, wherein SEQ ID NO 105 is a clone designatedherein as “DNA84141-2556”.

FIG. 106 shows the amino acid sequence (SEQ ID NO:106) derived from thecoding sequence of SEQ ID NO:105 shown in FIG. 105.

FIG. 107 shows a nucleotide sequence (SEQ ID NO:107) of a nativesequence PRO4404 cDNA, wherein SEQ ID NO:107 is a clone designatedherein as “DNA84142-2613”.

FIG. 108 shows the amino acid sequence (SEQ ID NO:108) derived from thecoding sequence of SEQ ID NO:107 shown in FIG. 107.

FIG. 109 shows a nucleotide sequence (SEQ ID NO:109) of a nativesequence PRO1884 cDNA, wherein SEQ ID NO:109 is a clone designatedherein as “DNA84318-2520”.

FIG. 110 shows the amino acid sequence (SEQ ID NO:110) derived from thecoding sequence of SEQ ID NO:109 shown in FIG. 109.

FIG. 111 shows a nucleotide sequence (SEQ ID NO:111) of a nativesequence PRO4349 cDNA, wherein SEQ ID NO:111 is a clone designatedherein as “DNA84909-2590”.

FIG. 112 shows the amino acid sequence (SEQ ID NO:112) derived from thecoding sequence of SEQ ID NO:111 shown in FIG. 111.

FIG. 113 shows a nucleotide sequence (SEQ ID NO:113) of a nativesequence PRO4401 cDNA, wherein SEQ ID NO:113 is a clone designatedherein as “DNA84912-2610”.

FIG. 114 shows the amino acid sequence (SEQ ID NO:114) derived from thecoding sequence of SEQ ID NO:113 shown in FIG. 113.

FIG. 115 shows a nucleotide sequence (SEQ ID NO:115) of a nativesequence PRO1867 cDNA, wherein SEQ ID NO:115 is a clone designatedherein as “DNA84925-2514”.

FIG. 116 shows the amino acid sequence (SEQ ID NO:116) derived from thecoding sequence of SEQ ID NO:115 shown in FIG. 115.

FIG. 117 shows a nucleotide sequence (SEQ ID NO:117) of a nativesequence PRO4319 cDNA, wherein SEQ ID NO:117 is a clone designatedherein as “DNA84928-2564”.

FIG. 118 shows the amino acid sequence (SEQ ID NO:118) derived from thecoding sequence of SEQ ID NO:117 shown in FIG. 117.

FIG. 119 shows a nucleotide sequence (SEQ ID NO:119) of a nativesequence PRO4991 cDNA, wherein SEQ ID NO:119 is a clone designatedherein as “DNA84932-2657”.

FIG. 120 shows the amino acid sequence (SEQ ID NO:120) derived from thecoding sequence of SEQ ID NO:119 shown in FIG. 119.

FIG. 121 shows a nucleotide sequence (SEQ ID NO:121) of a nativesequence PRO4398 cDNA, wherein SEQ ID NO:121 is a clone designatedherein as “DNA86592-2607”.

FIG. 122 shows the amino acid sequence (SEQ ID NO:122) derived from thecoding sequence of SEQ ID NO:121 shown in FIG. 121.

FIG. 123 shows a nucleotide sequence (SEQ ID NO:123) of a nativesequence PRO4346 cDNA, wherein SEQ ID NO:123 is a clone designatedherein as “DNA86594-2587”.

FIG. 124 shows the amino acid sequence (SEQ ID NO:124) derived from thecoding sequence of SEQ ID NO:123 shown in FIG. 123.

FIG. 125 shows a nucleotide sequence (SEQ ID NO:125) of a nativesequence PRO4350 cDNA, wherein SEQ ID NO:125 is a clone designatedherein as “DNA86647-2591”.

FIG. 126 shows the amino acid sequence (SEQ ID NO:126) derived from thecoding sequence of SEQ ID NO:125 shown in FIG. 125.

FIG. 127 shows a nucleotide sequence (SEQ ID NO:127) of a nativesequence PRO4318 cDNA, wherein SEQ ID NO:127 is a clone designatedherein as “DNA87185-2563”.

FIG. 128 shows the amino acid sequence (SEQ ID NO:128) derived from thecoding sequence of SEQ ID NO:127 shown in FIG. 127.

FIG. 129 shows a nucleotide sequence (SEQ ID NO:129) of a nativesequence PRO4340 cDNA, wherein SEQ ID NO:129 is a clone designatedherein as “DNA87656-2582”.

FIG. 130 shows the amino acid sequence (SEQ ID NO:130) derived from thecoding sequence of SEQ ID NO:129 shown in FIG. 129.

FIG. 131 shows a nucleotide sequence (SEQ ID NO:131) of a nativesequence PRO4400 cDNA, wherein SEQ ID NO:131 is a clone designatedherein as “DNA87974-2609”.

FIG. 132 shows the amino acid sequence (SEQ ID NO:132) derived from thecoding sequence of SEQ ID NO:131 shown in FIG. 131.

FIG. 133 shows a nucleotide sequence (SEQ ID NO:133) of a nativesequence PRO4320 cDNA, wherein SEQ ID NO:133 is a clone designatedherein as “DNA88001-2565”.

FIG. 134 shows the amino acid sequence (SEQ ID NO:134) derived from thecoding sequence of SEQ ID NO:133 shown in FIG. 133.

FIG. 135 shows a nucleotide sequence (SEQ ID NO:135) of a nativesequence PRO4409 cDNA, wherein SEQ ID NO:135 is a clone designatedherein as “DNA88004-2575”.

FIG. 136 shows the amino acid sequence (SEQ ID NO:136) derived from thecoding sequence of SEQ ID NO:135 shown in FIG. 135.

FIG. 137 shows a nucleotide sequence (SEQ ID NO:137) of a nativesequence PRO4399 cDNA, wherein SEQ ID NO:137 is a clone designatedherein as “DNA89220-2608”.

FIG. 138 shows the amino acid sequence (SEQ ID NO:138) derived from thecoding sequence of SEQ ID NO:137 shown in FIG. 137.

FIG. 139 shows a nucleotide sequence (SEQ ID NO:139) of a nativesequence PRO4418 cDNA, wherein SEQ ID NO:139 is a clone designatedherein as “DNA89947-2618”.

FIG. 140 shows the amino acid sequence (SEQ ID NO:140) derived from thecoding sequence of SEQ ID NO:139 shown in FIG. 139.

FIG. 141 shows a nucleotide sequence (SEQ ID NO:141) of a nativesequence PRO4330 cDNA, wherein SEQ ID NO:141 is a clone designatedherein as “DNA90842-2574”.

FIG. 142 shows the amino acid sequence (SEQ ID NO:142) derived from thecoding sequence of SEQ ID NO:141 shown in FIG. 141.

FIG. 143 shows a nucleotide sequence (SEQ ID NO:143) of a nativesequence PRO4339 cDNA, wherein SEQ ID NO:143 is a clone designatedherein as “DNA91775-2581”.

FIG. 144 shows the amino acid sequence (SEQ ID NO:144) derived from thecoding sequence of SEQ ID NO:143 shown in FIG. 143.

FIG. 145 shows a nucleotide sequence (SEQ ID NO:145) of a nativesequence PRO4326 cDNA, wherein SEQ ID NO:145 is a clone designatedherein as “DNA91779-2571”.

FIG. 146 shows the amino acid sequence (SEQ ID NO:146) derived from thecoding sequence of SEQ ID NO:145 shown in FIG. 145.

FIG. 147 shows a nucleotide sequence (SEQ ID NO:147) of a nativesequence PRO6014 cDNA, wherein SEQ ID NO:147 is a clone designatedherein as “DNA92217-2697”.

FIG. 148 shows the amino acid sequence (SEQ ID NO:148) derived from thecoding sequence of SEQ ID NO:147 shown in FIG. 147.

FIG. 149 shows a nucleotide sequence (SEQ ID NO:149) of a nativesequence PRO3446 cDNA, wherein SEQ ID NO:149 is a clone designatedherein as “DNA92219-2541”.

FIG. 150 shows the amino acid sequence (SEQ ID NO:150) derived from thecoding sequence of SEQ ID NO:149 shown in FIG. 149.

FIG. 151 shows a nucleotide sequence (SEQ ID NO:151) of a nativesequence PRO4322 cDNA, wherein SEQ ID NO:151 is a clone designatedherein as “DNA92223-2567”.

FIG. 152 shows the amino acid sequence (SEQ ID NO:152) derived from thecoding sequence of SEQ ID NO:151 shown in FIG. 151.

FIG. 153 shows a nucleotide sequence (SEQ ID NO:153) of a nativesequence PRO4381 cDNA, wherein SEQ ID NO:153 is a clone designatedherein as “DNA92225-2603”.

FIG. 154 shows the amino acid sequence (SEQ ID NO:154) derived from thecoding sequence of SEQ ID NO:153 shown in FIG. 153.

FIG. 155 shows a nucleotide sequence (SEQ ID NO:155) of a nativesequence PRO4348 cDNA, wherein SEQ ID NO:155 is a clone designatedherein as “DNA92232-2589”.

FIG. 156 shows the amino acid sequence (SEQ ID NO:156) derived from thecoding sequence of SEQ ID NO:155 shown in FIG. 155.

FIG. 157 shows a nucleotide sequence (SEQ ID NO:157) of a nativesequence PRO4371 cDNA, wherein SEQ ID NO:157 is a clone designatedherein as “DNA92233-2599”.

FIG. 158 shows the amino acid sequence (SEQ ID NO:158) derived from thecoding sequence of SEQ ID NO:157 shown in FIG. 157.

FIG. 159 shows a nucleotide sequence (SEQ ID NO:159) of a nativesequence PRO3742 cDNA, wherein SEQ ID NO:159 is a clone designatedherein as “DNA92243-2549”.

FIG. 160 shows the amino acid sequence (SEQ ID NO:160) derived from thecoding sequence of SEQ ID NO:159 shown in FIG. 159.

FIG. 161 shows a nucleotide sequence (SEQ ID NO:161) of a nativesequence PRO5773 cDNA, wherein SEQ ID NO:161 is a clone designatedherein as “DNA92253-2671”.

FIG. 162 shows the amino acid sequence (SEQ ID NO:162) derived from thecoding sequence of SEQ ID NO:161 shown in FIG. 161.

FIG. 163 shows a nucleotide sequence (SEQ ID NO:163) of a nativesequence PRO5774 cDNA, wherein SEQ ID NO:163 is a clone designatedherein as “DNA92254-2672”.

FIG. 164 shows the amino acid sequence (SEQ ID NO:164) derived from thecoding sequence of SEQ ID NO:163 shown in FIG. 163.

FIG. 165 shows a nucleotide sequence (SEQ ID NO:165) of a nativesequence PRO4343 cDNA, wherein SEQ ID NO:165 is a clone designatedherein as “DNA92255-2584”.

FIG. 166 shows the amino acid sequence (SEQ ID NO:166) derived from thecoding sequence of SEQ ID NO:165 shown in FIG. 165.

FIG. 167 shows a nucleotide sequence (SEQ ID NO:167) of a nativesequence PRO4325 cDNA, wherein SEQ ID NO:167 is a clone designatedherein as “DNA92269-2570”.

FIG. 168 shows the amino acid sequence (SEQ ID NO:168) derived from thecoding sequence of SEQ ID NO:167 shown in FIG. 167.

FIG. 169 shows a nucleotide sequence (SEQ ID NO:169) of a nativesequence PRO4347 cDNA, wherein SEQ ID NO:169 is a clone designatedherein as “DNA92288-2588”.

FIG. 170 shows the amino acid sequence (SEQ ID NO:170) derived from thecoding sequence of SEQ ID NO:169 shown in FIG. 169.

FIG. 171 shows a nucleotide sequence (SEQ ID NO:171) of a nativesequence PRO3743 cDNA, wherein SEQ ID NO:171 is a clone designatedherein as “DNA92290-2550”.

FIG. 172 shows the amino acid sequence (SEQ ID NO:172) derived from thecoding sequence of SEQ ID NO:171 shown in FIG. 171.

FIG. 173 shows a nucleotide sequence (SEQ ID NO:173) of a nativesequence PRO4426 cDNA, wherein SEQ ID NO:173 is a clone designatedherein as “DNA93012-2622”.

FIG. 174 shows the amino acid sequence (SEQ ID NO:174) derived from thecoding sequence of SEQ ID NO:173 shown in FIG. 173.

FIG. 175 shows a nucleotide sequence (SEQ ID NO:175) of a nativesequence PRO4500 cDNA, wherein SEQ ID NO:175 is a clone designatedherein as “DNA93020-2642”.

FIG. 176 shows the amino acid sequence (SEQ ID NO:176) derived from thecoding sequence of SEQ ID NO:175 shown in FIG. 175.

FIG. 177 shows a nucleotide sequence (SEQ ID NO:177) of a nativesequence PRO4389 cDNA, wherein SEQ ID NO:177 is a clone designatedherein as “DNA94830-2604”.

FIG. 178 shows the amino acid sequence (SEQ ID NO:178) derived from thecoding sequence of SEQ ID NO:177 shown in FIG. 177.

FIG. 179 shows a nucleotide sequence (SEQ ID NO:179) of a nativesequence PRO4337 cDNA, wherein SEQ ID NO:179 is a clone designatedherein as “DNA94833-2579”.

FIG. 180 shows the amino acid sequence (SEQ ID NO: 180) derived from thecoding sequence of SEQ ID NO:179 shown in FIG. 179.

FIG. 181 shows a nucleotide sequence (SEQ ID NO:181) of a nativesequence PRO4992 cDNA, wherein SEQ ID NO:181 is a clone designatedherein as “DNA94838-2658”.

FIG. 182 shows the amino acid sequence (SEQ ID NO:182) derived from thecoding sequence of SEQ ID NO:181 shown in FIG. 181.

FIG. 183 shows a nucleotide sequence (SEQ ID NO:183) of a nativesequence PRO5996 cDNA, wherein SEQ ID NO:183 is a clone designatedherein as “DNA94844-2686”.

FIG. 184 shows the amino acid sequence (SEQ ID NO:184) derived from thecoding sequence of SEQ ID NO:183 shown in FIG. 183.

FIG. 185 shows a nucleotide sequence (SEQ ID NO:185) of a nativesequence PRO4345 cDNA, wherein SEQ ID NO:185 is a clone designatedherein as “DNA94854-2586”.

FIG. 186 shows the amino acid sequence (SEQ ID NO:186) derived from thecoding sequence of SEQ ID NO:185 shown in FIG. 185.

FIG. 187 shows a nucleotide sequence (SEQ ID NO:187) of a nativesequence PRO4978 cDNA, wherein SEQ ID NO:187 is a clone designatedherein as “DNA95930”.

FIG. 188 shows the amino acid sequence (SEQ ID NO:188) derived from thecoding sequence of SEQ ID NO:187 shown in FIG. 187.

FIG. 189 shows a nucleotide sequence (SEQ ID NO:189) of a nativesequence PRO5780 cDNA, wherein SEQ ID NO:189 is a clone designatedherein as “DNA96868-2677”.

FIG. 190 shows the amino acid sequence (SEQ ID NO:190) derived from thecoding sequence of SEQ ID NO:189 shown in FIG. 189.

FIG. 191 shows a nucleotide sequence (SEQ ID NO:191) of a nativesequence PRO5992 cDNA, wherein SEQ ID NO:191 is a clone designatedherein as “DNA96871-2683”.

FIG. 192 shows the amino acid sequence (SEQ ID NO:192) derived from thecoding sequence of SEQ ID NO:191 shown in FIG. 191.

FIG. 193 shows a nucleotide sequence (SEQ ID NO:193) of a nativesequence PRO4428 cDNA, wherein SEQ ID NO:193 is a clone designatedherein as “DNA96880-2624”.

FIG. 194 shows the amino acid sequence (SEQ ID NO:194) derived from thecoding sequence of SEQ ID NO:193 shown in FIG. 193.

FIG. 195 shows a nucleotide sequence (SEQ ID NO:195) of a nativesequence PRO4994 cDNA, wherein SEQ ID NO:195 is a clone designatedherein as “DNA96986-2660”.

FIG. 196 shows the amino acid sequence (SEQ ID NO:196) derived from thecoding sequence of SEQ ID NO:195 shown in FIG. 195.

FIG. 197 shows a nucleotide sequence (SEQ ID NO:197) of a nativesequence PRO5995 cDNA, wherein SEQ ID NO:197 is a clone designatedherein as “DNA96988-2685”.

FIG. 198 shows the amino acid sequence (SEQ ID NO:198) derived from thecoding sequence of SEQ ID NO:197 shown in FIG. 197.

FIG. 199 shows a nucleotide sequence (SEQ ID NO:199) of a nativesequence PRO6094 cDNA, wherein SEQ ID NO:199 is a clone designatedherein as “DNA96995-2709”.

FIG. 200 shows the amino acid sequence (SEQ ID NO:200) derived from thecoding sequence of SEQ ID NO:199 shown in FIG. 199.

FIG. 201 shows a nucleotide sequence (SEQ ID NO:201) of a nativesequence PRO4317 cDNA, wherein SEQ ID NO:201 is a clone designatedherein as “DNA97004-2562”.

FIG. 202 shows the amino acid sequence (SEQ ID NO:202) derived from thecoding sequence of SEQ ID NO:201 shown in FIG. 201.

FIG. 203 shows a nucleotide sequence (SEQ ID NO:203) of a nativesequence PRO5997 cDNA, wherein SEQ ID NO:203 is a clone designatedherein as “DNA97005-2687”.

FIG. 204 shows the amino acid sequence (SEQ ID NO:204) derived from thecoding sequence of SEQ ID NO:203 shown in FIG. 203.

FIG. 205 shows a nucleotide sequence (SEQ ID NO:205) of a nativesequence PRO5005 cDNA, wherein SEQ ID NO:205 is a clone designatedherein as “DNA97009-2668”.

FIG. 206 shows the amino acid sequence (SEQ ID NO:206) derived from thecoding sequence of SEQ ID NO:205 shown in FIG. 205.

FIG. 207 shows a nucleotide sequence (SEQ ID NO:207) of a nativesequence PRO5004 cDNA, wherein SEQ ID NO:207 is a clone designatedherein as “DNA97013-2667”.

FIG. 208 shows the amino acid sequence (SEQ ID NO:208) derived from thecoding sequence of SEQ ID NO:207 shown in FIG. 207.

FIG. 209 shows a nucleotide sequence (SEQ ID NO:209) of a nativesequence PRO6001 cDNA, wherein SEQ ID NO:209 is a clone designatedherein as “DNA98380-2690”.

FIG. 210 shows the amino acid sequence (SEQ ID NO:210) derived from thecoding sequence of SEQ ID NO:209 shown in FIG. 209.

FIG. 211 shows a nucleotide sequence (SEQ ID NO:211) of a nativesequence PRO6013 cDNA, wherein SEQ ID NO:211 is a clone designatedherein as “DNA98561-2696”.

FIG. 212 shows the amino acid sequence (SEQ ID NO:212) derived from thecoding sequence of SEQ ID NO:211 shown in FIG. 211.

FIG. 213 shows a nucleotide sequence (SEQ ID NO:213) of a nativesequence PRO4502 cDNA, wherein SEQ ID NO:213 is a clone designatedherein as “DNA98575-2644”.

FIG. 214 shows the amino acid sequence (SEQ ID NO:214) derived from thecoding sequence of SEQ ID NO:213 shown in FIG. 213.

FIG. 215 shows a nucleotide sequence (SEQ ID NO:215) of a nativesequence PRO6007 cDNA, wherein SEQ ID NO:215 is a clone designatedherein as “DNA98593-2694”.

FIG. 216 shows the amino acid sequence (SEQ ID NO:216) derived from thecoding sequence of SEQ ID NO:215 shown in FIG. 215.

FIG. 217 shows a nucleotide sequence (SEQ ID NO:217) of a nativesequence PRO6028 cDNA, wherein SEQ ID NO:217 is a clone designatedherein as “DNA98600-2703”.

FIG. 218 shows the amino acid sequence (SEQ ID NO:218) derived from thecoding sequence of SEQ ID NO:217 shown in FIG. 217.

FIG. 219 shows a nucleotide sequence (SEQ ID NO:219) of a nativesequence PRO100 cDNA, wherein SEQ ID NO:219 is a clone designated hereinas “DNA99333”.

FIG. 220 shows the amino acid sequence (SEQ ID NO:220) derived from thecoding sequence of SEQ ID NO:219 shown in FIG. 219.

FIG. 221 shows a nucleotide sequence (SEQ ID NO:221) of a nativesequence PRO4327 cDNA, wherein SEQ ID NO:221 is a clone designatedherein as “DNA99391-2572”.

FIG. 222 shows the amino acid sequence (SEQ ID NO:222) derived from thecoding sequence of SEQ ID NO:221 shown in FIG. 221.

FIG. 223 shows a nucleotide sequence (SEQ ID NO:223) of a nativesequence PRO4315 cDNA, wherein SEQ ID NO:223 is a clone designatedherein as “DNA99393-2560”.

FIG. 224 shows the amino acid sequence (SEQ ID NO:224) derived from thecoding sequence of SEQ ID NO:223 shown in FIG. 223.

FIG. 225 shows a nucleotide sequence (SEQ ID NO:225) of a nativesequence PRO5993 cDNA, wherein SEQ ID NO:225 is a clone designatedherein as “DNA100276-2684”.

FIG. 226 shows the amino acid sequence (SEQ ID NO:226) derived from thecoding sequence of SEQ ID NO:225 shown in FIG. 225.

FIG. 227 shows a nucleotide sequence (SEQ ID NO:227) of a nativesequence PRO4503 cDNA, wherein SEQ ID NO:227 is a clone designatedherein as “DNA100312-2645”.

FIG. 228 shows the amino acid sequence (SEQ ID NO:228) derived from thecoding sequence of SEQ ID NO:227 shown in FIG. 227.

FIG. 229 shows a nucleotide sequence (SEQ ID NO:229) of a nativesequence PRO4976 cDNA, wherein SEQ ID NO:229 is a clone designatedherein as “DNA100902-2646”.

FIG. 230 shows the amino acid sequence (SEQ ID NO:230) derived from thecoding sequence of SEQ ID NO:229 shown in FIG. 229.

FIG. 231 shows a nucleotide sequence (SEQ ID NO:231) of a nativesequence PRO5798 cDNA, wherein SEQ ID NO:231 is a clone designatedherein as “DNA102899-2679”.

FIG. 232 shows the amino acid sequence (SEQ ID NO:232) derived from thecoding sequence of SEQ ID NO:231 shown in FIG. 231.

FIG. 233 shows a nucleotide sequence (SEQ ID NO:233) of a nativesequence PRO6242 cDNA, wherein SEQ ID NO:233 is a clone designatedherein as “DNA104875-2720”.

FIG. 234 shows the amino acid sequence (SEQ ID NO:234) derived from thecoding sequence of SEQ ID NO:233 shown in FIG. 233.

FIG. 235 shows a nucleotide sequence (SEQ ID NO:235) of a nativesequence PRO6095 cDNA, wherein SEQ ID NO:235 is a clone designatedherein as “DNA105680-2710”.

FIG. 236 shows the amino acid sequence (SEQ ID NO:236) derived from thecoding sequence of SEQ ID NO:235 shown in FIG. 235.

FIG. 237 shows a nucleotide sequence (SEQ ID NO:237) of a nativesequence PRO6093 cDNA, wherein SEQ ID NO:237 is a clone designatedherein as “DNA105779-2708”.

FIG. 238 shows the amino acid sequence (SEQ ID NO:238) derived from thecoding sequence of SEQ ID NO:237 shown in FIG. 237.

FIG. 239 shows a nucleotide sequence (SEQ ID NO:239) of a nativesequence PRO6012 cDNA, wherein SEQ ID NO:239 is a clone designatedherein as “DNA105794-2695”.

FIG. 240 shows the amino acid sequence (SEQ ID NO:240) derived from thecoding sequence of SEQ ID NO:239 shown in FIG. 239.

FIG. 241 shows a nucleotide sequence (SEQ ID NO:241) of a nativesequence PRO6027 cDNA, wherein SEQ ID NO:241 is a clone designatedherein as “DNA105838-2702”.

FIG. 242 shows the amino acid sequence (SEQ ID NO:242) derived from thecoding sequence of SEQ ID NO:241 shown in FIG. 241.

FIG. 243 shows a nucleotide sequence (SEQ ID NO:243) of a nativesequence PRO6181 cDNA, wherein SEQ ID NO:243 is a clone designatedherein as “DNA107698-2715”.

FIG. 244 shows the amino acid sequence (SEQ ID NO:244) derived from thecoding sequence of SEQ ID NO:243 shown in FIG. 243.

FIG. 245 shows a nucleotide sequence (SEQ ID NO:245) of a nativesequence PRO6097 cDNA, wherein SEQ ID NO:245 is a clone designatedherein as “DNA107701-2711”.

FIG. 246 shows the amino acid sequence (SEQ ID NO:246) derived from thecoding sequence of SEQ ID NO:245 shown in FIG. 245.

FIG. 247 shows a nucleotide sequence (SEQ ID NO:247) of a nativesequence PRO6090 cDNA, wherein SEQ ID NO:247 is a clone designatedherein as “DNA107781-2707”.

FIG. 248 shows the amino acid sequence (SEQ ID NO:248) derived from thecoding sequence of SEQ ID NO:247 shown in FIG. 247.

FIG. 249 shows a nucleotide sequence (SEQ ID NO:249) of a nativesequence PRO7171 cDNA, wherein SEQ ID NO:249 is a clone designatedherein as “DNA108670-2744”.

FIG. 250 shows the amino acid sequence (SEQ ID NO:250) derived from thecoding sequence of SEQ ID NO:249 shown in FIG. 249.

FIG. 251 shows a nucleotide sequence (SEQ ID NO:251) of a nativesequence PRO6258 cDNA, wherein SEQ ID NO:251 is a clone designatedherein as “DNA108688-2725”.

FIG. 252 shows the amino acid sequence (SEQ ID NO:252) derived from thecoding sequence of SEQ ID NO:251 shown in FIG. 251.

FIG. 253 shows a nucleotide sequence (SEQ ID NO:253) of a nativesequence PRO9820 cDNA, wherein SEQ ID NO:253 is a clone designatedherein as “DNA108769-2765”.

FIG. 254 shows the amino acid sequence (SEQ ID NO:254) derived from thecoding sequence of SEQ ID NO:253 shown in FIG. 253.

FIG. 255 shows a nucleotide sequence (SEQ ID NO:255) of a nativesequence PRO6243 cDNA, wherein SEQ ID NO:255 is a clone designatedherein as “DNA108935-2721”.

FIG. 256 shows the amino acid sequence (SEQ ID NO:256) derived from thecoding sequence of SEQ ID NO:255 shown in FIG. 255.

FIG. 257 shows a nucleotide sequence (SEQ ID NO:257) of a nativesequence PRO6182 cDNA, wherein SEQ ID NO:257 is a clone designatedherein as “DNA110700-2716”.

FIG. 258 shows the amino acid sequence (SEQ ID NO:258) derived from thecoding sequence of SEQ ID NO:257 shown in FIG. 257.

FIG. 259 shows a nucleotide sequence (SEQ ID NO:259) of a nativesequence PRO6079 cDNA, wherein SEQ ID NO:259 is a clone designatedherein as “DNA 11750-2706”.

FIG. 260 shows the amino acid sequence (SEQ ID NO:260) derived from thecoding sequence of SEQ ID NO:259 shown in FIG. 259.

FIG. 261 shows a nucleotide sequence (SEQ ID NO:261) of a nativesequence PRO7434 cDNA, wherein SEQ ID NO:261 is a clone designatedherein as “DNA123430-2755”.

FIG. 262 shows the amino acid sequence (SEQ ID NO:262) derived from thecoding sequence of SEQ ID NO:261 shown in FIG. 261.

FIG. 263 shows a nucleotide sequence (SEQ ID NO:263) of a nativesequence PRO9865 cDNA, wherein SEQ ID NO:263 is a clone designatedherein as “DNA125154-2785”.

FIG. 264 shows the amino acid sequence (SEQ ID NO:264) derived from thecoding sequence of SEQ ID NO:263 shown in FIG. 263.

FIG. 265 shows a nucleotide sequence (SEQ ID NO:265) of a nativesequence PRO9828 cDNA, wherein SEQ ID NO:265 is a clone designatedherein as “DNA142238-2768”.

FIG. 266 shows the amino acid sequence (SEQ ID NO:266) derived from thecoding sequence of SEQ ID NO:265 shown in FIG. 265.

FIG. 267 shows a nucleotide sequence (SEQ ID NO:267) of a nativesequence PRO196 cDNA, wherein SEQ ID NO:267 is a clone designated hereinas “DNA22779-1130”.

FIG. 268 shows the amino acid sequence (SEQ ID NO:268) derived from thecoding sequence of SEQ ID NO:267 shown in FIG. 267.

FIG. 269 shows a nucleotide sequence (SEQ ID NO:269) of a nativesequence PRO197 cDNA, wherein SEQ ID NO:269 is a clone designated hereinas “DNA22780-1078”.

FIG. 270 shows the amino acid sequence (SEQ ID NO:270) derived from thecoding sequence of SEQ ID NO:269 shown in FIG. 269.

FIG. 271 shows a nucleotide sequence (SEQ ID NO:271) of a nativesequence PRO195 cDNA, wherein SEQ ID NO:271 is a clone designated hereinas “DNA26847-1395”.

FIG. 272 shows the amino acid sequence (SEQ ID NO:272) derived from thecoding sequence of SEQ ID NO:271 shown in FIG. 271.

FIG. 273 shows a nucleotide sequence (SEQ ID NO:273) of a nativesequence PRO187 cDNA, wherein SEQ ID NO:273 is a clone designated hereinas “DNA27864-1155”.

FIG. 274 shows the amino acid sequence (SEQ ID NO:274) derived from thecoding sequence of SEQ ID NO:273 shown in FIG. 273.

FIG. 275 shows a nucleotide sequence (SEQ ID NO:275) of a nativesequence PRO182 cDNA, wherein SEQ ID NO:275 is a clone designated hereinas “DNA27865-1091”.

FIG. 276 shows the amino acid sequence (SEQ ID NO:276) derived from thecoding sequence of SEQ ID NO:275 shown in FIG. 275.

FIG. 277 shows a nucleotide sequence (SEQ ID NO:277) of a nativesequence PRO188 cDNA, wherein SEQ ID NO:277 is a clone designated hereinas “DNA28497-1130”.

FIG. 278 shows the amino acid sequence (SEQ ID NO:278) derived from thecoding sequence of SEQ ID NO:277 shown in FIG. 277.

FIG. 279 shows a nucleotide sequence (SEQ ID NO:279) of a nativesequence PRO183 cDNA, wherein SEQ ID NO:279 is a clone designated hereinas “DNA28498”.

FIG. 280 shows the amino acid sequence (SEQ ID NO:280) derived from thecoding sequence of SEQ ID NO:279 shown in FIG. 279.

FIG. 281 shows a nucleotide sequence (SEQ ID NO:281) of a nativesequence PRO184 cDNA, wherein SEQ ID NO:281 is a clone designated hereinas “DNA28500”.

FIG. 282 shows the amino acid sequence (SEQ ID NO:282) derived from thecoding sequence of SEQ ID NO:281 shown in FIG. 281.

FIG. 283 shows a nucleotide sequence (SEQ ID NO:283) of a nativesequence PRO185 cDNA, wherein SEQ ID NO:283 is a clone designated hereinas “DNA28503”.

FIG. 284 shows the amino acid sequence (SEQ ID NO:284) derived from thecoding sequence of SEQ ID NO:283 shown in FIG. 283.

FIG. 285 shows a nucleotide sequence (SEQ ID NO:285) of a nativesequence PRO200 cDNA, wherein SEQ ID NO:285 is a clone designated hereinas “DNA29101-1122”.

FIG. 286 shows the amino acid sequence (SEQ ID NO:286) derived from thecoding sequence of SEQ ID NO:285 shown in FIG. 285.

FIG. 287 shows a nucleotide sequence (SEQ ID NO:287) of a nativesequence PRO202 cDNA, wherein SEQ ID NO:287 is a clone designated hereinas “DNA30869”.

FIG. 288 shows the amino acid sequence (SEQ ID NO:288) derived from thecoding sequence of SEQ ID NO:287 shown in FIG. 287.

FIG. 289 shows a nucleotide sequence (SEQ ID NO:289) of a nativesequence PRO214 cDNA, wherein SEQ ID NO:289 is a clone designated hereinas “DNA32286-1191”.

FIG. 290 shows the amino acid sequence (SEQ ID NO:290) derived from thecoding sequence of SEQ ID NO:289 shown in FIG. 289.

FIG. 291 shows a nucleotide sequence (SEQ ID NO:291) of a nativesequence PRO215 cDNA, wherein SEQ ID NO:291 is a clone designated hereinas “DNA32288-1132”.

FIG. 292 shows the amino acid sequence (SEQ ID NO:292) derived from thecoding sequence of SEQ ID NO:291 shown in FIG. 291.

FIG. 293 shows a nucleotide sequence (SEQ ID NO:293) of a nativesequence PRO219 cDNA, wherein SEQ ID NO:293 is a clone designated hereinas “DNA32290-1164”.

FIG. 294 shows the amino acid sequence (SEQ ID NO:294) derived from thecoding sequence of SEQ ID NO:293 shown in FIG. 293.

FIG. 295 shows a nucleotide sequence (SEQ ID NO:295) of a nativesequence PRO211 cDNA, wherein SEQ ID NO:295 is a clone designated hereinas “DNA32292-1131”.

FIG. 296 shows the amino acid sequence (SEQ ID NO:296) derived from thecoding sequence of SEQ ID NO:295 shown in FIG. 295.

FIG. 297 shows a nucleotide sequence (SEQ ID NO:297) of a nativesequence PRO220 cDNA, wherein SEQ ID NO:297 is a clone designated hereinas “DNA32298-1132”.

FIG. 298 shows the amino acid sequence (SEQ ID NO:298) derived from thecoding sequence of SEQ ID NO:297 shown in FIG. 297.

FIG. 299 shows a nucleotide sequence (SEQ ID NO:299) of a nativesequence PRO366 cDNA, wherein SEQ ID NO:299 is a clone designated hereinas “DNA33085-1110”.

FIG. 300 shows the amino acid sequence (SEQ ID NO:300) derived from thecoding sequence of SEQ ID NO:299 shown in FIG. 299.

FIG. 301 shows a nucleotide sequence (SEQ ID NO:301) of a nativesequence PRO216 cDNA, wherein SEQ ID NO:301 is a clone designated hereinas “DNA33087-1158”.

FIG. 302 shows the amino acid sequence (SEQ ID NO:302) derived from thecoding sequence of SEQ ID NO:301 shown in FIG. 301.

FIG. 303 shows a nucleotide sequence (SEQ ID NO:303) of a nativesequence PRO221 cDNA, wherein SEQ ID NO:303 is a clone designated hereinas “DNA33089-1132”.

FIG. 304 shows the amino acid sequence (SEQ ID NO:304) derived from thecoding sequence of SEQ ID NO:303 shown in FIG. 303.

FIG. 305 shows a nucleotide sequence (SEQ ID NO:305) of a nativesequence PRO228 cDNA, wherein SEQ ID NO:305 is a clone designated hereinas “DNA33092-1202”.

FIG. 306 shows the amino acid sequence (SEQ ID NO:306) derived from thecoding sequence of SEQ ID NO:305 shown in FIG. 305.

FIG. 307 shows a nucleotide sequence (SEQ ID NO:307) of a nativesequence PRO217 cDNA, wherein SEQ ID NO:307 is a clone designated hereinas “DNA33094-1131 ”.

FIG. 308 shows the amino acid sequence (SEQ ID NO:308) derived from thecoding sequence of SEQ ID NO:307 shown in FIG. 307.

FIG. 309 shows a nucleotide sequence (SEQ ID NO:309) of a nativesequence PRO222 cDNA, wherein SEQ ID NO:309 is a clone designated hereinas “DNA33107-1135”.

FIG. 310 shows the amino acid sequence (SEQ ID NO:310) derived from thecoding sequence of SEQ ID NO:309 shown in FIG. 309.

FIG. 311 shows a nucleotide sequence (SEQ ID NO:311) of a nativesequence PRO224 cDNA, wherein SEQ ID NO:311 is a clone designated hereinas “DNA33221-1133”.

FIG. 312 shows the amino acid sequence (SEQ ID NO:312) derived from thecoding sequence of SEQ ID NO:311 shown in FIG. 311.

FIG. 313 shows a nucleotide sequence (SEQ ID NO:313) of a nativesequence PRO230 cDNA, wherein SEQ ID NO:313 is a clone designated hereinas “DNA33223-1136”.

FIG. 314 shows the amino acid sequence (SEQ ID NO:314) derived from thecoding sequence of SEQ ID NO:313 shown in FIG. 313.

FIG. 315 shows a nucleotide sequence (SEQ ID NO:315) of a nativesequence PRO198 cDNA, wherein SEQ ID NO:315 is a clone designated hereinas “DNA33457-1078”.

FIG. 316 shows the amino acid sequence (SEQ ID NO:316) derived from thecoding sequence of SEQ ID NO:315 shown in FIG. 315.

FIG. 317 shows a nucleotide sequence (SEQ ID NO:317) of a nativesequence PRO226 cDNA, wherein SEQ ID NO:317 is a clone designated hereinas “DNA33460-1166”.

FIG. 318 shows the amino acid sequence (SEQ ID NO:318) derived from thecoding sequence of SEQ ID NO:317 shown in FIG. 317.

FIG. 319 shows a nucleotide sequence (SEQ ID NO:319) of a nativesequence PRO261 cDNA, wherein SEQ ID NO:319 is a clone designated hereinas “DNA33473-1176”.

FIG. 320 shows the amino acid sequence (SEQ ID NO:320) derived from thecoding sequence of SEQ ID NO:319 shown in FIG. 319.

FIG. 321 shows a nucleotide sequence (SEQ ID NO:321) of a nativesequence PRO242 cDNA, wherein SEQ ID NO:321 is a clone designated hereinas “DNA33785-1143”.

FIG. 322 shows the amino acid sequence (SEQ ID NO:322) derived from thecoding sequence of SEQ ID NO:321 shown in FIG. 321.

FIG. 323 shows a nucleotide sequence (SEQ ID NO:323) of a nativesequence PRO227 cDNA, wherein SEQ ID NO:323 is a clone designated hereinas “DNA33786-1132”.

FIG. 324 shows the amino acid sequence (SEQ ID NO:324) derived from thecoding sequence of SEQ ID NO:323 shown in FIG. 323.

FIG. 325 shows a nucleotide sequence (SEQ ID NO:325) of a nativesequence PRO237 cDNA, wherein SEQ ID NO:325 is a clone designated hereinas “DNA34353-1428”.

FIG. 326 shows the amino acid sequence (SEQ ID NO:326) derived from thecoding sequence of SEQ ID NO:325 shown in FIG. 325.

FIG. 327 shows a nucleotide sequence (SEQ ID NO:327) of a nativesequence PRO241 cDNA, wherein SEQ ID NO:327 is a clone designated hereinas “DNA34392-1170”.

FIG. 328 shows the amino acid sequence (SEQ ID NO:328) derived from thecoding sequence of SEQ ID NO:327 shown in FIG. 327.

FIG. 329 shows a nucleotide sequence (SEQ ID NO:329) of a nativesequence PRO231 cDNA, wherein SEQ ID NO:329 is a clone designated hereinas “DNA34434-1139”.

FIG. 330 shows the amino acid sequence (SEQ ID NO:330) derived from thecoding sequence of SEQ ID NO:329 shown in FIG. 329.

FIG. 331 shows a nucleotide sequence (SEQ ID NO:331) of a nativesequence PRO235 cDNA, wherein SEQ ID NO:331 is a clone designated hereinas “DNA35558-1167”.

FIG. 332 shows the amino acid sequence (SEQ ID NO:332) derived from thecoding sequence of SEQ ID NO:331 shown in FIG. 331.

FIG. 333 shows a nucleotide sequence (SEQ ID NO:333) of a nativesequence PRO323 cDNA, wherein SEQ ID NO:333 is a clone designated hereinas “DNA35595-1228”.

FIG. 334 shows the amino acid sequence (SEQ ID NO:334) derived from thecoding sequence of SEQ ID NO:333 shown in FIG. 333.

FIG. 335 shows a nucleotide sequence (SEQ ID NO:335) of a nativesequence PRO245 cDNA, wherein SEQ ID NO:335 is a clone designated hereinas “DNA35638-1216”.

FIG. 336 shows the amino acid sequence (SEQ ID NO:336) derived from thecoding sequence of SEQ ID NO:335 shown in FIG. 335.

FIG. 337 shows a nucleotide sequence (SEQ ID NO:337) of a nativesequence PRO246 cDNA, wherein SEQ ID NO:337 is a clone designated hereinas “DNA35639-1172”.

FIG. 338 shows the amino acid sequence (SEQ ID NO:338) derived from thecoding sequence of SEQ ID NO:337 shown in FIG. 337.

FIG. 339 shows a nucleotide sequence (SEQ ID NO:339) of a nativesequence PRO288 cDNA, wherein SEQ ID NO:339 is a clone designated hereinas “DNA35663-1129”.

FIG. 340 shows the amino acid sequence (SEQ ID NO:340) derived from thecoding sequence of SEQ ID NO:339 shown in FIG. 339.

FIG. 341 shows a nucleotide sequence (SEQ ID NO:341) of a nativesequence PRO248 cDNA, wherein SEQ ID NO:341 is a clone designated hereinas “DNA35674-1142”.

FIG. 342 shows the amino acid sequence (SEQ ID NO:342) derived from thecoding sequence of SEQ ID NO:341 shown in FIG. 341.

FIG. 343 shows a nucleotide sequence (SEQ ID NO:343) of a nativesequence PRO257 cDNA, wherein SEQ ID NO:343 is a clone designated hereinas “DNA35841-1173”.

FIG. 344 shows the amino acid sequence (SEQ ID NO:344) derived from thecoding sequence of SEQ ID NO:343 shown in FIG. 343.

FIG. 345 shows a nucleotide sequence (SEQ ID NO:345) of a nativesequence PRO172 cDNA, wherein SEQ ID NO:345 is a clone designated hereinas “DNA35916-1161”.

FIG. 346 shows the amino acid sequence (SEQ ID NO:346) derived from thecoding sequence of SEQ ID NO:345 shown in FIG. 345.

FIG. 347 shows a nucleotide sequence (SEQ ID NO:347) of a nativesequence PRO258 cDNA, wherein SEQ ID NO:347 is a clone designated hereinas “DNA35918-1174”.

FIG. 348 shows the amino acid sequence (SEQ ID NO:348) derived from thecoding sequence of SEQ ID NO:347 shown in FIG. 347.

FIG. 349 shows a nucleotide sequence (SEQ ID NO:349) of a nativesequence PRO265 cDNA, wherein SEQ ID NO:349 is a clone designated hereinas “DNA36350-1158”.

FIG. 350 shows the amino acid sequence (SEQ ID NO:350) derived from thecoding sequence of SEQ ID NO:349 shown in FIG. 349.

FIG. 351 shows a nucleotide sequence (SEQ ID NO:351) of a nativesequence PRO326 cDNA, wherein SEQ ID NO:351 is a clone designated hereinas “DNA37140-1234”.

FIG. 352 shows the amino acid sequence (SEQ ID NO:352) derived from thecoding sequence of SEQ ID NO:351 shown in FIG. 351.

FIG. 353 shows a nucleotide sequence (SEQ ID NO:353) of a nativesequence PRO266 cDNA, wherein SEQ ID NO:353 is a clone designated hereinas “DNA37150-1178”.

FIG. 354 shows the amino acid sequence (SEQ ID NO:354) derived from thecoding sequence of SEQ ID NO:353 shown in FIG. 353.

FIG. 355 shows a nucleotide sequence (SEQ ID NO:355) of a nativesequence PRO269 cDNA, wherein SEQ ID NO:355 is a clone designated hereinas “DNA38260-1180”.

FIG. 356 shows the amino acid sequence (SEQ ID NO:356) derived from thecoding sequence of SEQ ID NO:355 shown in FIG. 355.

FIG. 357 shows a nucleotide sequence (SEQ ID NO:357) of a nativesequence PRO285 cDNA, wherein SEQ ID NO:357 is a clone designated hereinas “DNA40021-1154”.

FIG. 358 shows the amino acid sequence (SEQ ID NO:358) derived from thecoding sequence of SEQ ID NO:357 shown in FIG. 357.

FIG. 359 shows a nucleotide sequence (SEQ ID NO:359) of a nativesequence PRO328 cDNA, wherein SEQ ID NO:359 is a clone designated hereinas “DNA40587-1231”.

FIG. 360 shows the amino acid sequence (SEQ ID NO:360) derived from thecoding sequence of SEQ ID NO:359 shown in FIG. 359.

FIG. 361 shows a nucleotide sequence (SEQ ID NO:361) of a nativesequence PRO344 cDNA, wherein SEQ ID NO:361 is a clone designated hereinas “DNA40592-1242”.

FIG. 362 shows the amino acid sequence (SEQ ID NO:362) derived from thecoding sequence of SEQ ID NO:361 shown in FIG. 361.

FIG. 363 shows a nucleotide sequence (SEQ ID NO:363) of a nativesequence PRO272 cDNA, wherein SEQ ID NO:363 is a clone designated hereinas “DNA40620-1183”.

FIG. 364 shows the amino acid sequence (SEQ ID NO:364) derived from thecoding sequence of SEQ ID NO:363 shown in FIG. 363.

FIG. 365 shows a nucleotide sequence (SEQ ID NO:365) of a nativesequence PRO301 cDNA, wherein SEQ ID NO:365 is a clone designated hereinas “DNA40628-1216”.

FIG. 366 shows the amino acid sequence (SEQ ID NO:366) derived from thecoding sequence of SEQ ID NO:365 shown in FIG. 365.

FIG. 367 shows a nucleotide sequence (SEQ ID NO:367) of a nativesequence PRO331 cDNA, wherein SEQ ID NO:367 is a clone designated hereinas “DNA40981-1234”.

FIG. 368 shows the amino acid sequence (SEQ ID NO:368) derived from thecoding sequence of SEQ ID NO:367 shown in FIG. 367.

FIG. 369 shows a nucleotide sequence (SEQ ID NO:369) of a nativesequence PRO332 cDNA, wherein SEQ ID NO:369 is a clone designated hereinas “DNA40982-1235”.

FIG. 370 shows the amino acid sequence (SEQ ID NO:370) derived from thecoding sequence of SEQ ID NO:369 shown in FIG. 369.

FIG. 371 shows a nucleotide sequence (SEQ ID NO:371) of a nativesequence PRO353 cDNA, wherein SEQ ID NO:371 is a clone designated hereinas “DNA41234-1242”.

FIG. 372 shows the amino acid sequence (SEQ ID NO:372) derived from thecoding sequence of SEQ ID NO:371 shown in FIG. 371.

FIG. 373 shows a nucleotide sequence (SEQ ID NO:373) of a nativesequence PRO310 cDNA, wherein SEQ ID NO:373 is a clone designated hereinas “DNA43046-1225”.

FIG. 374 shows the amino acid sequence (SEQ ID NO:374) derived from thecoding sequence of SEQ ID NO:373 shown in FIG. 373.

FIG. 375 shows a nucleotide sequence (SEQ ID NO:375) of a nativesequence PRO337 cDNA, wherein SEQ ID NO:375 is a clone designated hereinas “DNA43316-1237”.

FIG. 376 shows the amino acid sequence (SEQ ID NO:376) derived from thecoding sequence of SEQ ID NO:375 shown in FIG. 375.

FIG. 377 shows a nucleotide sequence (SEQ ID NO:377) of a nativesequence PRO346 cDNA, wherein SEQ ID NO:377 is a clone designated hereinas “DNA44167-1243”.

FIG. 378 shows the amino acid sequence (SEQ ID NO:378) derived from thecoding sequence of SEQ ID NO:377 shown in FIG. 377.

FIG. 379 shows a nucleotide sequence (SEQ ID NO:379) of a nativesequence PRO350 cDNA, wherein SEQ ID NO:379 is a clone designated hereinas “DNA44175-1314”.

FIG. 380 shows the amino acid sequence (SEQ ID NO:380) derived from thecoding sequence of SEQ ID NO:379 shown in FIG. 379.

FIG. 381 shows a nucleotide sequence (SEQ ID NO:381) of a nativesequence PRO526 cDNA, wherein SEQ ID NO:381 is a clone designated hereinas “DNA44184-1319”.

FIG. 382 shows the amino acid sequence (SEQ ID NO:382) derived from thecoding sequence of SEQ ID NO:381 shown in FIG. 381.

FIG. 383 shows a nucleotide sequence (SEQ ID NO:383) of a nativesequence PRO381 cDNA, wherein SEQ ID NO:383 is a clone designated hereinas “DNA44194-1317”.

FIG. 384 shows the amino acid sequence (SEQ ID NO:384) derived from thecoding sequence of SEQ ID NO:383 shown in FIG. 383.

FIG. 385 shows a nucleotide sequence (SEQ ID NO:385) of a nativesequence PRO846 cDNA, wherein SEQ ID NO:385 is a clone designated hereinas “DNA44196-1353”.

FIG. 386 shows the amino acid sequence (SEQ ID NO:386) derived from thecoding sequence of SEQ ID NO:385 shown in FIG. 385.

FIG. 387 shows a nucleotide sequence (SEQ ID NO:387) of a nativesequence PRO363 cDNA, wherein SEQ ID NO:387 is a clone designated hereinas “DNA45419-1252”.

FIG. 388 shows the amino acid sequence (SEQ ID NO:388) derived from thecoding sequence of SEQ ID NO:387 shown in FIG. 387.

FIG. 389 shows a nucleotide sequence (SEQ ID NO:389) of a nativesequence PRO365 cDNA, wherein SEQ ID NO:389 is a clone designated hereinas “DNA46777-1253”.

FIG. 390 shows the amino acid sequence (SEQ ID NO:390) derived from thecoding sequence of SEQ ID NO:389 shown in FIG. 389.

FIG. 391 shows a nucleotide sequence (SEQ ID NO:391) of a nativesequence PRO1310 cDNA, wherein SEQ ID NO:391 is a clone designatedherein as “DNA47394-1572”.

FIG. 392 shows the amino acid sequence (SEQ ID NO:392) derived from thecoding sequence of SEQ ID NO:391 shown in FIG. 391.

FIG. 393 shows a nucleotide sequence (SEQ ID NO:393) of a nativesequence PRO731 cDNA, wherein SEQ ID NO:393 is a clone designated hereinas “DNA48331-1329”.

FIG. 394 shows the amino acid sequence (SEQ ID NO:394) derived from thecoding sequence of SEQ ID NO:393 shown in FIG. 393.

FIG. 395 shows a nucleotide sequence (SEQ ID NO:395) of a nativesequence PRO322 cDNA, wherein SEQ ID NO:395 is a clone designated hereinas “DNA48336-1309”.

FIG. 396 shows the amino acid sequence (SEQ ID NO:396) derived from thecoding sequence of SEQ ID NO:395 shown in FIG. 395.

FIG. 397 shows a nucleotide sequence (SEQ ID NO:397) of a nativesequence PRO536 cDNA, wherein SEQ ID NO:397 is a clone designated hereinas “DNA49142-1430”.

FIG. 398 shows the amino acid sequence (SEQ ID NO:398) derived from thecoding sequence of SEQ ID NO:397 shown in FIG. 397.

FIG. 399 shows a nucleotide sequence (SEQ ID NO:399) of a nativesequence PRO719 cDNA, wherein SEQ ID NO:399 is a clone designated hereinas “DNA49646-1327”.

FIG. 400 shows the amino acid sequence (SEQ ID NO:400) derived from thecoding sequence of SEQ ID NO:399 shown in FIG. 399.

FIG. 401 shows a nucleotide sequence (SEQ ID NO:401) of a nativesequence PRO619 cDNA, wherein SEQ ID NO:401 is a clone designated hereinas “DNA49821-1562”.

FIG. 402 shows the amino acid sequence (SEQ ID NO:402) derived from thecoding sequence of SEQ ID NO:401 shown in FIG. 401.

FIG. 403 shows a nucleotide sequence (SEQ ID NO:403) of a nativesequence PRO771 cDNA, wherein SEQ ID NO:403 is a clone designated hereinas “DNA49829-1346”.

FIG. 404 shows the amino acid sequence (SEQ ID NO:404) derived from thecoding sequence of SEQ ID NO:403 shown in FIG. 403.

FIG. 405 shows a nucleotide sequence (SEQ ID NO:405) of a nativesequence PRO1083 cDNA, wherein SEQ ID NO:405 is a clone designatedherein as “DNA50921-1458”.

FIG. 406 shows the amino acid sequence (SEQ ID NO:406) derived from thecoding sequence of SEQ ID NO:405 shown in FIG. 405.

FIG. 407 shows a nucleotide sequence (SEQ ID NO:407) of a nativesequence PRO862 cDNA, wherein SEQ ID NO:407 is a clone designated hereinas “DNA52187-1354”.

FIG. 408 shows the amino acid sequence (SEQ ID NO:408) derived from thecoding sequence of SEQ ID NO:407 shown in FIG. 407.

FIG. 409 shows a nucleotide sequence (SEQ ID NO:409) of a nativesequence PRO733 cDNA, wherein SEQ ID NO:409 is a clone designated hereinas “DNA52196-1348”.

FIG. 410 shows the amino acid sequence (SEQ ID NO:410) derived from thecoding sequence of SEQ ID NO:409 shown in FIG. 409.

FIG. 411 shows a nucleotide sequence (SEQ ID NO:411) of a nativesequence PRO1188 cDNA, wherein SEQ ID NO:411 is a clone designatedherein as “DNA52598-1518”.

FIG. 412 shows the amino acid sequence (SEQ ID NO:412) derived from thecoding sequence of SEQ ID NO:411 shown in FIG. 411.

FIG. 413 shows a nucleotide sequence (SEQ ID NO:413) of a nativesequence PRO770 cDNA, wherein SEQ ID NO:413 is a clone designated hereinas “DNA54228-1366”.

FIG. 414 shows the amino acid sequence (SEQ ID NO:414) derived from thecoding sequence of SEQ ID NO:413 shown in FIG. 413.

FIG. 415 shows a nucleotide sequence (SEQ ID NO:415) of a nativesequence PRO1080 cDNA, wherein SEQ ID NO:415 is a clone designatedherein as “DNA56047-1456”.

FIG. 416 shows the amino acid sequence (SEQ ID NO:416) derived from thecoding sequence of SEQ ID NO:415 shown in FIG. 415.

FIG. 417 shows a nucleotide sequence (SEQ ID NO:417) of a nativesequence PRO1017 cDNA, wherein SEQ ID NO:417 is a clone designatedherein as “DNA56112-1379”.

FIG. 418 shows the amino acid sequence (SEQ ID NO:418) derived from thecoding sequence of SEQ ID NO:417 shown in FIG. 417.

FIG. 419 shows a nucleotide sequence (SEQ ID NO:419) of a nativesequence PRO1016 cDNA, wherein SEQ ID NO:419 is a clone designatedherein as “DNA56113-1378”.

FIG. 420 shows the amino acid sequence (SEQ ID NO:420) derived from thecoding sequence of SEQ ID NO:419 shown in FIG. 419.

FIG. 421 shows a nucleotide sequence (SEQ ID NO:421) of a nativesequence PRO792 cDNA, wherein SEQ ID NO:421 is a clone designated hereinas “DNA56352-1358”.

FIG. 422 shows the amino acid sequence (SEQ ID NO:422) derived from thecoding sequence of SEQ ID NO:421 shown in FIG. 421.

FIG. 423 shows a nucleotide sequence (SEQ ID NO:423) of a nativesequence PRO938 cDNA, wherein SEQ ID NO:423 is a clone designated hereinas “DNA56433-1406”.

FIG. 424 shows the amino acid sequence (SEQ ID NO:424) derived from thecoding sequence of SEQ ID NO:423 shown in FIG. 423.

FIG. 425 shows a nucleotide sequence (SEQ ID NO:425) of a nativesequence PRO1012 cDNA, wherein SEQ ID NO:425 is a clone designatedherein as “DNA56439-1376”.

FIG. 426 shows the amino acid sequence (SEQ ID NO:426) derived from thecoding sequence of SEQ ID NO:425 shown in FIG. 425.

FIG. 427 shows a nucleotide sequence (SEQ ID NO:427) of a nativesequence PRO1008 cDNA, wherein SEQ ID NO:427 is a clone designatedherein as “DNA57530-1375”.

FIG. 428 shows the amino acid sequence (SEQ ID NO:428) derived from thecoding sequence of SEQ ID NO:427 shown in FIG. 427.

FIG. 429 shows a nucleotide sequence (SEQ ID NO:429) of a nativesequence PRO1075 cDNA, wherein SEQ ID NO:429 is a clone designatedherein as “DNA57689-1385”.

FIG. 430 shows the amino acid sequence (SEQ ID NO:430) derived from thecoding sequence of SEQ ID NO:429 shown in FIG. 429.

FIG. 431 shows a nucleotide sequence (SEQ ID NO:431) of a nativesequence PRO1007 cDNA, wherein SEQ ID NO:431 is a clone designatedherein as “DNA57690-1374”.

FIG. 432 shows the amino acid sequence (SEQ ID NO:432) derived from thecoding sequence of SEQ ID NO:431 shown in FIG. 431.

FIG. 433 shows a nucleotide sequence (SEQ ID NO:433) of a nativesequence PRO1056 cDNA, wherein SEQ ID NO:433 is a clone designatedherein as “DNA57693-1424”.

FIG. 434 shows the amino acid sequence (SEQ ID NO:434) derived from thecoding sequence of SEQ ID NO:433 shown in FIG. 433.

FIG. 435 shows a nucleotide sequence (SEQ ID NO:435) of a nativesequence PRO791 cDNA, wherein SEQ ID NO:435 is a clone designated hereinas “DNA57838-1337”.

FIG. 436 shows the amino acid sequence (SEQ ID NO:436) derived from thecoding sequence of SEQ ID NO:435 shown in FIG. 435.

FIG. 437 shows a nucleotide sequence (SEQ ID NO:437) of a nativesequence PRO1111 cDNA, wherein SEQ ID NO:437 is a clone designatedherein as “DNA58721-1475”.

FIG. 438 shows the amino acid sequence (SEQ ID NO:438) derived from thecoding sequence of SEQ ID NO:437 shown in FIG. 437.

FIG. 439 shows a nucleotide sequence (SEQ ID NO:439) of a nativesequence PRO812 cDNA, wherein SEQ ID NO:439 is a clone designated hereinas “DNA59205-1421”.

FIG. 440 shows the amino acid sequence (SEQ ID NO:440) derived from thecoding sequence of SEQ ID NO:439 shown in FIG. 439.

FIG. 441 shows a nucleotide sequence (SEQ ID NO:441) of a nativesequence PRO1066 cDNA, wherein SEQ ID NO:441 is a clone designatedherein as “DNA59215-1425”.

FIG. 442 shows the amino acid sequence (SEQ ID NO:442) derived from thecoding sequence of SEQ ID NO:441 shown in FIG. 441.

FIG. 443 shows a nucleotide sequence (SEQ ID NO:443) of a nativesequence PRO1185 cDNA, wherein SEQ ID NO:443 is a clone designatedherein as “DNA59220-1514”.

FIG. 444 shows the amino acid sequence (SEQ ID NO:444) derived from thecoding sequence of SEQ ID NO:443 shown in FIG. 443.

FIG. 445 shows a nucleotide sequence (SEQ ID NO:445) of a nativesequence PRO1031 cDNA, wherein SEQ ID NO:445 is a clone designatedherein as “DNA59294-1381”.

FIG. 446 shows the amino acid sequence (SEQ ID NO:446) derived from thecoding sequence of SEQ ID NO:445 shown in FIG. 445.

FIG. 447 shows a nucleotide sequence (SEQ ID NO:447) of a nativesequence PRO1360 cDNA, wherein SEQ ID NO:447 is a clone designatedherein as “DNA59488-1603”.

FIG. 448 shows the amino acid sequence (SEQ ID NO:448) derived from thecoding sequence of SEQ ID NO:447 shown in FIG. 447.

FIG. 449 shows a nucleotide sequence (SEQ ID NO:449) of a nativesequence PRO1309 cDNA, wherein SEQ ID NO:449 is a clone designatedherein as “DNA59588-1571”.

FIG. 450 shows the amino acid sequence (SEQ ID NO:450) derived from thecoding sequence of SEQ ID NO:449 shown in FIG. 449.

FIG. 451 shows a nucleotide sequence (SEQ ID NO:451) of a nativesequence PRO1107 cDNA, wherein SEQ ID NO:451 is a clone designatedherein as “DNA59606-1471”.

FIG. 452 shows the amino acid sequence (SEQ ID NO:452) derived from thecoding sequence of SEQ ID NO:451 shown in FIG. 451.

FIG. 453 shows a nucleotide sequence (SEQ ID NO:453) of a nativesequence PRO836 cDNA, wherein SEQ ID NO:453 is a clone designated hereinas “DNA59620-1463”.

FIG. 454 shows the amino acid sequence (SEQ ID NO:454) derived from thecoding sequence of SEQ ID NO:453 shown in FIG. 453.

FIG. 455 shows a nucleotide sequence (SEQ ID NO:455) of a nativesequence PRO1132 cDNA, wherein SEQ ID NO:455 is a clone designatedherein as “DNA59767-1489”.

FIG. 456 shows the amino acid sequence (SEQ ID NO:456) derived from thecoding sequence of SEQ ID NO:455 shown in FIG. 455.

FIG. 457 shows a nucleotide sequence (SEQ ID NO:457) of a nativesequence PRO1131 cDNA, wherein SEQ ID NO:457 is a clone designatedherein as “DNA59777-1480”.

FIG. 458 shows the amino acid sequence (SEQ ID NO:458) derived from thecoding sequence of SEQ ID NO:457 shown in FIG. 457.

FIG. 459 shows a nucleotide sequence (SEQ ID NO:459) of a nativesequence PRO1130 cDNA, wherein SEQ ID NO:459 is a clone designatedherein as “DNA59814-1486”.

FIG. 460 shows the amino acid sequence (SEQ ID NO:460) derived from thecoding sequence of SEQ ID NO:459 shown in FIG. 459.

FIG. 461 shows a nucleotide sequence (SEQ ID NO:461) of a nativesequence PRO844 cDNA, wherein SEQ ID NO:461 is a clone designated hereinas “DNA59839-1461”.

FIG. 462 shows the amino acid sequence (SEQ ID NO:462) derived from thecoding sequence of SEQ ID NO:461 shown in FIG. 461.

FIG. 463 shows a nucleotide sequence (SEQ ID NO:463) of a nativesequence PRO1154 cDNA, wherein SEQ ID NO:463 is a clone designatedherein as “DNA59846-1503”.

FIG. 464 shows the amino acid sequence (SEQ ID NO:464) derived from thecoding sequence of SEQ ID NO:463 shown in FIG. 463.

FIG. 465 shows a nucleotide sequence (SEQ ID NO:465) of a nativesequence PRO1181 cDNA, wherein SEQ ID NO:465 is a clone designatedherein as “DNA59847-1511”.

FIG. 466 shows the amino acid sequence (SEQ ID NO:466) derived from thecoding sequence of SEQ ID NO:465 shown in FIG. 465.

FIG. 467 shows a nucleotide sequence (SEQ ID NO:467) of a nativesequence PRO1126 cDNA, wherein SEQ ID NO:467 is a clone designatedherein as “DNA60615-1483”.

FIG. 468 shows the amino acid sequence (SEQ ID NO:468) derived from thecoding sequence of SEQ ID NO:467 shown in FIG. 467.

FIG. 469 shows a nucleotide sequence (SEQ ID NO:469) of a nativesequence PRO1186 cDNA, wherein SEQ ID NO:469 is a clone designatedherein as “DNA60621-1516”.

FIG. 470 shows the amino acid sequence (SEQ ID NO:470) derived from thecoding sequence of SEQ ID NO:469 shown in FIG. 469.

FIG. 471 shows a nucleotide sequence (SEQ ID NO:471) of a nativesequence PRO1198 cDNA, wherein SEQ ID NO:471 is a clone designatedherein as “DNA60622-1525”.

FIG. 472 shows the amino acid sequence (SEQ ID NO:472) derived from thecoding sequence of SEQ ID NO:471 shown in FIG. 471.

FIG. 473 shows a nucleotide sequence (SEQ ID NO:473) of a nativesequence PRO1159 cDNA, wherein SEQ ID NO:473 is a clone designatedherein as “DNA60627-1508”.

FIG. 474 shows the amino acid sequence (SEQ ID NO:474) derived from thecoding sequence of SEQ ID NO:473 shown in FIG. 473.

FIG. 475 shows a nucleotide sequence (SEQ ID NO:475) of a nativesequence PRO1265 cDNA, wherein SEQ ID NO:475 is a clone designatedherein as “DNA60764-1533”.

FIG. 476 shows the amino acid sequence (SEQ ID NO:476) derived from thecoding sequence of SEQ ID NO:475 shown in FIG. 475.

FIG. 477 shows a nucleotide sequence (SEQ ID NO:477) of a nativesequence PRO1250 cDNA, wherein SEQ ID NO:477 is a clone designatedherein as “DNA60775-1532”.

FIG. 478 shows the amino acid sequence (SEQ ID NO:478) derived from thecoding sequence of SEQ ID NO:477 shown in FIG. 477.

FIG. 479 shows a nucleotide sequence (SEQ ID NO:479) of a nativesequence PRO1475 cDNA, wherein SEQ ID NO:479 is a clone designatedherein as “DNA61185-1646”.

FIG. 480 shows the amino acid sequence (SEQ ID NO:480) derived from thecoding sequence of SEQ ID NO:479 shown in FIG. 479.

FIG. 481 shows a nucleotide sequence (SEQ ID NO:481) of a nativesequence PRO1312 cDNA, wherein SEQ ID NO:481 is a clone designatedherein as “DNA61873-1574”.

FIG. 482 shows the amino acid sequence (SEQ ID NO:482) derived from thecoding sequence of SEQ ID NO:481 shown in FIG. 481.

FIG. 483 shows a nucleotide sequence (SEQ ID NO:483) of a nativesequence PRO1308 cDNA, wherein SEQ ID NO:483 is a clone designatedherein as “DNA62306-1570”.

FIG. 484 shows the amino acid sequence (SEQ ID NO:484) derived from thecoding sequence of SEQ ID NO:483 shown in FIG. 483.

FIG. 485 shows a nucleotide sequence (SEQ ID NO:485) of a nativesequence PRO1326 cDNA, wherein SEQ ID NO:485 is a clone designatedherein as “DNA62808-1582”.

FIG. 486 shows the amino acid sequence (SEQ ID NO:486) derived from thecoding sequence of SEQ ID NO:485 shown in FIG. 485.

FIG. 487 shows a nucleotide sequence (SEQ ID NO:487) of a nativesequence PRO1192 cDNA, wherein SEQ ID NO:487 is a clone designatedherein as “DNA62814-1521”.

FIG. 488 shows the amino acid sequence (SEQ ID NO:488) derived from thecoding sequence of SEQ ID NO:487 shown in FIG. 487.

FIG. 489 shows a nucleotide sequence (SEQ ID NO:489) of a nativesequence PRO1246 cDNA, wherein SEQ ID NO:489 is a clone designatedherein as “DNA64885-1529”.

FIG. 490 shows the amino acid sequence (SEQ ID NO:490) derived from thecoding sequence of SEQ ID NO:489 shown in FIG. 489.

FIG. 491 shows a nucleotide sequence (SEQ ID NO:491) of a nativesequence PRO1356 cDNA, wherein SEQ ID NO:491 is a clone designatedherein as “DNA64886-1601”.

FIG. 492 shows the amino acid sequence (SEQ ID NO:492) derived from thecoding sequence of SEQ ID NO:491 shown in FIG. 491.

FIG. 493 shows a nucleotide sequence (SEQ ID NO:493) of a nativesequence PRO1275 cDNA, wherein SEQ ID NO:493 is a clone designatedherein as “DNA64888-1542”.

FIG. 494 shows the amino acid sequence (SEQ ID NO:494) derived from thecoding sequence of SEQ ID NO:493 shown in FIG. 493.

FIG. 495 shows a nucleotide sequence (SEQ ID NO:495) of a nativesequence PRO1274 cDNA, wherein SEQ ID NO:495 is a clone designatedherein as “DNA64889-1541”.

FIG. 496 shows the amino acid sequence (SEQ ID NO:496) derived from thecoding sequence of SEQ ID NO:495 shown in FIG. 495.

FIG. 497 shows a nucleotide sequence (SEQ ID NO:497) of a nativesequence PRO1358 cDNA, wherein SEQ ID NO:497 is a clone designatedherein as “DNA64890-1612”.

FIG. 498 shows the amino acid sequence (SEQ ID NO:498) derived from thecoding sequence of SEQ ID NO:497 shown in FIG. 497.

FIG. 499 shows a nucleotide sequence (SEQ ID NO:499) of a nativesequence PRO1286 cDNA, wherein SEQ ID NO:499 is a clone designatedherein as “DNA64903-1553”.

FIG. 500 shows the amino acid sequence (SEQ ID NO:500) derived from thecoding sequence of SEQ ID NO:499 shown in FIG. 499.

FIG. 501 shows a nucleotide sequence (SEQ ID NO:501) of a nativesequence PRO1294 cDNA, wherein SEQ ID NO:501 is a clone designatedherein as “DNA64905-1558”.

FIG. 502 shows the amino acid sequence (SEQ ID NO:502) derived from thecoding sequence of SEQ ID NO:501 shown in FIG. 501.

FIG. 503 shows a nucleotide sequence (SEQ ID NO:503) of a nativesequence PRO1273 cDNA, wherein SEQ ID NO:503 is a clone designatedherein as “DNA65402-1540”.

FIG. 504 shows the amino acid sequence (SEQ ID NO:504) derived from thecoding sequence of SEQ ID NO:503 shown in FIG. 503.

FIG. 505 shows a nucleotide sequence (SEQ ID NO:505) of a nativesequence PRO1279 cDNA, wherein SEQ ID NO:505 is a clone designatedherein as “DNA65405-1547”.

FIG. 506 shows the amino acid sequence (SEQ ID NO:506) derived from thecoding sequence of SEQ ID NO:505 shown in FIG. 505.

FIG. 507 shows a nucleotide sequence (SEQ ID NO:507) of a nativesequence PRO1195 cDNA, wherein SEQ ID NO:507 is a clone designatedherein as “DNA65412-1523”.

FIG. 508 shows the amino acid sequence (SEQ ID NO:508) derived from thecoding sequence of SEQ ID NO:507 shown in FIG. 507.

FIG. 509 shows a nucleotide sequence (SEQ ID NO:509) of a nativesequence PRO1271 cDNA, wherein SEQ ID NO:509 is a clone designatedherein as “DNA66309-1538”.

FIG. 510 shows the amino acid sequence (SEQ ID NO:510) derived from thecoding sequence of SEQ ID NO:509 shown in FIG. 509.

FIG. 511 shows a nucleotide sequence (SEQ ID NO:511) of a nativesequence PRO1338 cDNA, wherein SEQ ID NO:511 is a clone designatedherein as “DNA66667-1596”.

FIG. 512 shows the amino acid sequence (SEQ ID NO:512) derived from thecoding sequence of SEQ ID NO:511 shown in FIG. 511.

FIG. 513 shows a nucleotide sequence (SEQ ID NO:513) of a nativesequence PRO1343 cDNA, wherein SEQ ID NO:513 is a clone designatedherein as “DNA66675-1587”.

FIG. 514 shows the amino acid sequence (SEQ ID NO:514) derived from thecoding sequence of SEQ ID NO:513 shown in FIG. 513.

FIG. 515 shows a nucleotide sequence (SEQ ID NO:515) of a nativesequence PRO1434 cDNA, wherein SEQ ID NO:515 is a clone designatedherein as “DNA68818-2536”.

FIG. 516 shows the amino acid sequence (SEQ ID NO:516) derived from thecoding sequence of SEQ ID NO:515 shown in FIG. 515.

FIG. 517 shows a nucleotide sequence (SEQ ID NO:517) of a nativesequence PRO1418 cDNA, wherein SEQ ID NO:517 is a clone designatedherein as “DNA68864-1629”.

FIG. 518 shows the amino acid sequence (SEQ ID NO:518) derived from thecoding sequence of SEQ ID NO:517 shown in FIG. 517.

FIG. 519 shows a nucleotide sequence (SEQ ID NO:519) of a nativesequence PRO1387 cDNA, wherein SEQ ID NO:519 is a clone designatedherein as “DNA68872-1620”.

FIG. 520 shows the amino acid sequence (SEQ ID NO:520) derived from thecoding sequence of SEQ ID NO:519 shown in FIG. 519.

FIG. 521 shows a nucleotide sequence (SEQ ID NO:521) of a nativesequence PRO1384 cDNA, wherein SEQ ID NO:521 is a clone designatedherein as “DNA71159-1617”.

FIG. 522 shows the amino acid sequence (SEQ ID NO:522) derived from thecoding sequence of SEQ ID NO:521 shown in FIG. 521.

FIG. 523 shows a nucleotide sequence (SEQ ID NO:523) of a nativesequence PRO1565 cDNA, wherein SEQ ID NO:523 is a clone designatedherein as “DNA73727-1673”.

FIG. 524 shows the amino acid sequence (SEQ ID NO:524) derived from thecoding sequence of SEQ ID NO:523 shown in FIG. 523.

FIG. 525 shows a nucleotide sequence (SEQ ID NO:525) of a nativesequence PRO1474 cDNA, wherein SEQ ID NO:525 is a clone designatedherein as “DNA73739-1645”.

FIG. 526 shows the amino acid sequence (SEQ ID NO:526) derived from thecoding sequence of SEQ ID NO:525 shown in FIG. 525.

FIG. 527 shows a nucleotide sequence (SEQ ID NO:527) of a nativesequence PRO1917 cDNA, wherein SEQ ID NO:527 is a clone designatedherein as “DNA76400-2528”.

FIG. 528 shows the amino acid sequence (SEQ ID NO:528) derived from thecoding sequence of SEQ ID NO:527 shown in FIG. 527.

FIG. 529 shows a nucleotide sequence (SEQ ID NO:529) of a nativesequence PRO1787 cDNA, wherein SEQ ID NO:529 is a clone designatedherein as “DNA76510-2504”.

FIG. 530 shows the amino acid sequence (SEQ ID NO:530) derived from thecoding sequence of SEQ ID NO:529 shown in FIG. 529.

FIG. 531 shows a nucleotide sequence (SEQ ID NO:531) of a nativesequence PRO1556 cDNA, wherein SEQ ID NO:531 is a clone designatedherein as “DNA76529-1666”.

FIG. 532 shows the amino acid sequence (SEQ ID NO:532) derived from thecoding sequence of SEQ ID NO:531 shown in FIG. 531.

FIG. 533 shows a nucleotide sequence (SEQ ID NO:533) of a nativesequence PRO1561 cDNA, wherein SEQ ID NO:533 is a clone designatedherein as “DNA76538-1670”.

FIG. 534 shows the amino acid sequence (SEQ ID NO:534) derived from thecoding sequence of SEQ ID NO:533 shown in FIG. 533.

FIG. 535 shows a nucleotide sequence (SEQ ID NO:535) of a nativesequence PRO1693 cDNA, wherein SEQ ID NO:535 is a clone designatedherein as “DNA77301-1708”.

FIG. 536 shows the amino acid sequence (SEQ ID NO:536) derived from thecoding sequence of SEQ ID NO:535 shown in FIG. 535.

FIG. 537 shows a nucleotide sequence (SEQ ID NO:537) of a nativesequence PRO1868 cDNA, wherein SEQ ID NO:537 is a clone designatedherein as “DNA77624-2515”.

FIG. 538 shows the amino acid sequence (SEQ ID NO:538) derived from thecoding sequence of SEQ ID NO:537 shown in FIG. 537.

FIG. 539 shows a nucleotide sequence (SEQ ID NO:539) of a nativesequence PRO1890 cDNA, wherein SEQ ID NO:539 is a clone designatedherein as “DNA79230-2525”.

FIG. 540 shows the amino acid sequence (SEQ ID NO:540) derived from thecoding sequence of SEQ ID NO:539 shown in FIG. 539.

FIG. 541 shows a nucleotide sequence (SEQ ID NO:541) of a nativesequence PRO1887 cDNA, wherein SEQ ID NO:541 is a clone designatedherein as “DNA79862-2522”.

FIG. 542 shows the amino acid sequence (SEQ ID NO:542) derived from thecoding sequence of SEQ ID NO:541 shown in FIG. 541.

FIG. 543 shows a nucleotide sequence (SEQ ID NO:543) of a nativesequence PRO4353 cDNA, wherein SEQ ID NO:543 is a clone designatedherein as “DNA80145-2594”.

FIG. 544 shows the amino acid sequence (SEQ ID NO:544) derived from thecoding sequence of SEQ ID NO:543 shown in FIG. 543.

FIG. 545 shows a nucleotide sequence (SEQ ID NO:545) of a nativesequence PRO1801 cDNA, wherein SEQ ID NO:545 is a clone designatedherein as “DNA83500-2506”.

FIG. 546 shows the amino acid sequence (SEQ ID NO:546) derived from thecoding sequence of SEQ ID NO:545 shown in FIG. 545.

FIG. 547 shows a nucleotide sequence (SEQ ID NO:547) of a nativesequence PRO4357 cDNA, wherein SEQ ID NO:547 is a clone designatedherein as “DNA84917-2597”.

FIG. 548 shows the amino acid sequence (SEQ ID NO:548) derived from thecoding sequence of SEQ ID NO:547 shown in FIG. 547.

FIG. 549 shows a nucleotide sequence (SEQ ID NO:549) of a nativesequence PRO4302 cDNA, wherein SEQ ID NO:549 is a clone designatedherein as “DNA92218-2554”.

FIG. 550 shows the amino acid sequence (SEQ ID NO:550) derived from thecoding sequence of SEQ ID NO:549 shown in FIG. 549.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. Definitions

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods. Theterm “PRO polypeptide” refers to each individual PRO/number polypeptidedisclosed herein. All disclosures in this specification which refer tothe “PRO polypeptide” refer to each of the polypeptides individually aswell as jointly. For example, descriptions of the preparation of,purification of, derivation of, formation of antibodies to or against,administration of, compositions containing, treatment of a disease with,etc., pertain to each polypeptide of the invention individually. Theterm “PRO polypeptide” also includes variants of the PRO/numberpolypeptides disclosed herein.

A “native sequence PRO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding PRO polypeptide derivedfrom nature. Such native sequence PRO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PROpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

The PRO polypeptide “extracellular domain” or “ECD” refers to a form ofthe PRO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the PRO polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein. Optionally, therefore, an extracellular domain of aPRO polypeptide may contain from about 5 or fewer amino acids on eitherside of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are comtemplated by the present invention.

The approximate location of the “signal peptides” of the various PROpolypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10: 1–6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683–4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO polypeptide variant” means an active PRO polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, alternatively at least about 20 aminoacids in length, alternatively at least about 30 amino acids in length,alternatively at least about 40 amino acids in length, alternatively atleast about 50 amino acids in length, alternatively at least about 60amino acids in length, alternatively at least about 70 amino acids inlength, alternatively at least about 80 amino acids in length,alternatively at least about 90 amino acids in length, alternatively atleast about 100 amino acids in length, alternatively at least about 150amino acids in length, alternatively at least about 200 amino acids inlength, alternatively at least about 300 amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the PROpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389–3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be obtained from the National Institute of Health, Bethesda, Md.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff forfinal gapped alignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“PRO variant polynucleotide” or “PRO variant nucleic acid sequence”means a nucleic acid molecule which encodes an active PRO polypeptide asdefined below and which has at least about 80% nucleic acid sequenceidentity with a nucleotide acid sequence encoding a full-length nativesequence PRO polypeptide sequence as disclosed herein, a full-lengthnative sequence PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Ordinarily,a PRO variant polynucleotide will have at least about 80% nucleic acidsequence identity, alternatively at least about 81% nucleic acidsequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity with a nucleic acid sequence encoding a full-lengthnative sequence PRO polypeptide sequence as disclosed herein, afull-length native sequence PRO polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 120 nucleotides in length, alternatively atleast about 150 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 210 nucleotides inlength, alternatively at least about 240 nucleotides in length,alternatively at least about 270 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 600 nucleotides inlength, alternatively at least about 900 nucleotides in length, or more.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389–3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be obtained from the National Institute of Health, Bethesda, Md.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff forfinal gapped alignment25 and scoring matrix=BLOSUM62.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460–480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389–3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtainedfrom the National Institute of Health, Bethesda, Md. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO variant polynucleotides are nucleic acidmolecules that encode an active PRO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), anti-PRO antibodycompositions with polyepitopic specificity, single chain anti-PROantibodies, and fragments of anti-PRO antibodies (see below). The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and %SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37–50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of aPRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057–1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269–315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)–V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444–6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

An “effective amount” of a polypeptide disclosed herein or an agonist orantagonist thereof is an amount sufficient to carry out a specificallystated purpose. An “effective amount” may be determined empirically andin a routine manner, in relation to the stated purpose.

TABLE 1 /*  *  * C—C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop—stop = 0; J (joker)match = 0  */ #define _M −8 /* value of a match with a stop */ int_day[26][26] = { /*  A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */ {2, 0, −2, 0, 0, −4, 1, −1, −1, 0, −1, −2, −1, 0, _M, 1, 0,−2, 1, 1, 0, 0, −6, 0, −3, 0}, /* B */ {0, 3, −4, 3, 2, −5, 0, 1, −2, 0,0, −3, −2, 2, _M, −1, 1, 0, 0, 0, 0, −2, −5, 0, −3, 1}, /* C */ {−2, −4,15, −5, −5, −4, −3, −3, −2, 0, −5, −6, −5, −4, _M, −3, −5, −4, 0, −2, 0,−2, −8, 0, 0, −5}, /* D */ {0, 3, −5, 4, 3, −6, 1, 1, −2, 0, 0, −4, −3,2, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4, 2}, /* E */ {0, 2, −5, 3, 4,−5, 0, 1, −2, 0, 0, −3, −2, 1, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4,3}, /* F */ {−4, −5, −4, −6, −5, 9, −5, −2, 1, 0, −5, 2, 0, −4, _M, −5,−5, −4, −3, −3, 0, −1, 0, 0, 7, −5}, /* G */ {1, 0, −3, 1, 0, −5, 5, −2,−3, 0, −2, −4, −3, 0, _M, −1, −1, −3, 1, 0, 0, −1, −7, 0, −5, 0}, /* H*/ {−1, 1, −3, 1, 1, −2, −2, 6, −2, 0, 0, −2, −2, 2, _M, 0, 3, 2, −1,−1, 0, −2, −3, 0, 0, 2}, /* I */ {−1, −2, −2, −2, −2, 1, −3, −2, 5, 0,−2, 2, 2, −2, _M, −2, −2, −2, −1, 0, 0, 4, −5, 0, −1, −2}, /* J */ {0,0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,0}, /* K */ {−1, 0, −5, 0, 0, −5, −2, 0, −2, 0, 5, −3, 0, 1, _M, −1, 1,3, 0, 0, 0, −2, −3, 0, −4, 0}, /* L */ {−2, −3, −6, −4, −3, 2, −4, −2,2, 0, −3, 6, 4, −3, _M, −3, −2, −3, −3 , −1, 0, 2, −2, 0, −1, −2} /* M*/ {−1, −2, −5, −3, −2, 0, −3, −2, 2, 0, 0, 4, 6, −2, _M, −2, −1, 0, −2,−1, 0, 2, −4, 0, −2, −1}, /* N */ {0, 2, −4, 2, 1, −4, 0, 2, −2, 0, 1,−3, −2, 2, _M, −1, 1, 0, 1, 0, 0, −2, −4, 0, −2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,}, /* P */ {1, −1, −3, −1, −1, −5,−1, 0, −2, 0, −1, −3, −2, −1,_M, 6, 0, 0, 1, 0, 0, −1, −6, 0, −5, 0}, /*Q */ {0, 1, −5, 2, 2, −5, −1, 3, −2, 0, 1, −2, −1, 1, _M, 0, 4, 1, −1,−1, 0, −2, −5, 0, −4, 3}, /* R */ {−2, 0, −4, −1, −1, −4, −3, 2, −2, 0,3, −3, 0, 0, _M, 0, 1, 6, 0, −1, 0, −2, 2, 0, −4, 0}, /* S */ {1, 0, 0,0, 0, −3, 1, −1, −1, 0, 0, −3, −2, 1, _M, 1, −1, 0, 2, 1, 0, −1, −2, 0,−3, 0}, /* T */ {1, 0, −2, 0, 0, −3, 0, −1, 0, 0, 0, −1, −1, 0, _M, 0,−1, −1, 1, 3, 0, 0, −5, 0, −3, 0}, /* U */ {0, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {0, −2, −2,−2, −2, −1, −1, −2, 4, 0, −2, 2, 2, −2,_M, −1, −2, −2, −1, 0, 0, 4, −6,0, −2, −2}, /* W */ {−6, −5, −8, −7, −7, 0, −7, −3, −5, 0, −3, −2, −4,−4,_M, −6, −5, 2, −2, −5, 0, −6, 17, 0, 0, −6}, /* X */ {0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */{−3, −3, 0, −4, −4, 7, −5, 0, −1, 0, −4, −1, −2, −2, _M, −5, −4, −4, −3,−3, 0, −2, 0, 0, 10, −4}, /* Z */ {0, 1, −5, 2, 3, −5, 0, 2, −2, 0, 0,−2, −1, 1,_M, 0, 3, 0, 0, 0, 0, −2, −6, 0, −4, 4}, }; /*  */ #include<stdio.h> #include <ctype.h> #define MAXJMP  16 /* max jumps in a diag*/ #define MAXGAP  24 /* don't continue to penalize gaps larger thanthis */ #define JMPS 1024 /* max jmps in an path */ #define MX   4 /*save if there's at least MX-1 bases since last jmp */ #define DMAT   3/* value of matching bases */ #define DMIS   0 /* penalty for mismatchedbases */ #define DINS0   8 /* penalty for a gap */ #define DINS1   1 /*penalty per base */ #define PINS0   8 /* penalty for a gap */ #definePINS1   4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /*size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. ofjmp in seq x */ /* limits seq to 2{circumflex over ( )}16 −1 */ };struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for errmsgs */ char *seqx[2]; /* seqs: getseqs() */ int dmax; /* best diag:nw() */ int dmax0; /* final diag */ int dna; /* set if dna: main() */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw() */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char*getseq(), *g_calloc(); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  *  where file1 and file2 are two dna or twoprotein sequences.  *  The sequences can be in upper- or lower-case anmay contain ambiguity  *  Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  *  Max file length is 65535 (limited by unsigned short x in thejmp struct)  *  A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  *  Output is in the file “align.out”  *  * Theprogram may create a tmp file in /tmp to hold info about traceback.  *Original version developed under BSD 4.3 on a vax 8650  */ #include“nw.h” #include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1<<(‘D’–‘A’))|(1<<(‘N’–‘A’)), 4, 8, 16, 32, 64,128, 256, 0×FFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16, 1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22, 1<<23, 1<<24,1<<25|(1<<(‘E’–‘A’))|(1<<(‘Q’–‘A’)) }; main(ac, av) main int ac; char*av[]; { prog = av[0]; if(ac != 3) { fprintf(stderr, “usage: %s file1file2\n”, prog); fprintf(stderr, “where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr, “The sequences can be inupper- or lower-case\n”); fprintf(stderr, “Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr, “Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw(); /* fill in the matrix, getthe possible jmps */ readjmps(); /* get the actual jmps */ print(); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main()  * dna: values in Fitch andSmith, PNAS, 80, 1382–1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw() nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PlNS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0]−ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx = 0;} ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if(col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] =− ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy);if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps &&xx < len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) {smax = col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }(void) free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print() -- only routinevisible outside this module  *  * static:  * getmat() -- trace back bestpath, count matches: print()  * pr_align() -- print alignment ofdescribed in array p[]: print()  * dumpblock() -- dump a block of lineswith numbers, stars: pr_align()  * nums() -- put out a number line:dumpblock()  * putline() -- put out a line (name, [num], seq, [num]):dumpblock()  * stars() - -put a line of stars: dumpblock()  *stripname() -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC  3 #define P_LINE 256 /* maximum output line */#define P_SPC  3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print() print { int lx, ly, firstgap, lastgap;  /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr, “%s: can't write%s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1;firstgap = lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align(); } /*  * traceback the best path, count matches  */ static getmat(lx, ly, firstgap,lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap,lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1;char outx[32]; double pct; register n0, n1; register char *p0, *p1; /*get total matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (nl++ == pp[1].x[i1]) siz1 = pp[1].n[il++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); ps[i] = seqx[i]; po[i] = out[i]; } fprintf(fx, “<gaps infirst sequence: %d”, gapx); ...getmat if (gapx) { (void) sprintf(outx,“(%d %s%s)”, ngapx, (dna)? “base”: “residue”, (ngapx == 1)? “”:“s”);fprintf(fx, “%s”, outx); fprintf(fx, “, gaps in second sequence: %d”,gapy); if (gapy) { (void) sprintf(outx, “(%d %s%s)”, ngapy, (dna)?“base”:“residue”, (ngapy == 1)? “”:“s”); fprintf(fx, “%s”, outx); } if(dna) fprintf(fx, “\n<score: %d (match = %d, mismatch = %d, gap penalty= %d + %d per base)\n”, smax, DMAT, DMIS, DINS0, DINS1); elsefprintf(fx, “\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %dper residue)\n”, smax, PINS0, PINS1); if (endgaps) fprintf(fx, “<endgapspenalized. left endgap: %d %s%s, right endgap: %d %s%s\n”, firstgap,(dna)? “base” : “residue”, (firstgap == 1)? “” : “s”, lastgap, (dna)?“base” : “residue”, (lastgap == 1)? “” : “s”); else fprintf(fx,“<endgaps not penalized\n”); } static nm; /* matches in core -- forchecking */ static lmax; /* lengths of stripped file names */ staticij[2]; /* jmp index for a path */ static nc[2]; /* number at start ofcurrent line */ static ni[2]; /* current elem number -- for gapping */static siz[2]; static char *ps[2]; /* ptr to current element */ staticchar *po[2]; /* ptr to next output char slot */ static charout[2][P_LINE]; /* output line */ static char star[P_LINE]; /* set bystars() */ /*  * print alignment of described in struct path pp[]  */static pr_align() pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2;i++) { nn = stripname(namex[i]); if (nn >lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more;) {...pr_align for (i = more = 0; i < 2; i++) { /*  * do we have more ofthis sequence?  */ if (!*ps[i]) continue; more++; if (pp[i].spc) { /*leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if (siz[i]) { /* ina gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we're putting a seqelement */ *po[i] = *ps[i]; if (islower(*ps[i]))    *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] == pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock(); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align()  */ static dumpblock() dumpblock { register i; for(i =0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx); for(i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) != ‘’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars(); putline(i);if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } }/* * put out a number line: dumpblock()  */ static nums(ix) numsint  ix; /* index in out[] holding seq line */ { char nline[P_LINE];register i, j; register char *pn, *px, *py; for(pn = nline, i = 0; i <lmax+P_SPC; i++, pn++) *pn = ‘ ’; for (i = nc[ix], py = out[ix]; *py;py++, pn++) { if (*py == ‘ ’ || *py == ‘−’) *pn = ‘ ’; else { if (i%10== 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? −i : i; for (px = pn; j;j/= 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px = ‘−’; } else *pn = ‘ ’;i++; } } *pn = ‘\0’; nc[ix] = i; for (pn = nline; *pn; pn++) (void)putc(*pn, fx); (void) putc(‘\n’, fx); } /*  * put out a line (name,[num], seq. [num]): dumpblock()  */ static putline(ix) putline int   ix;{ ...putline int i; register char *px; for (px = namex[ix], i = 0; *px&& *px != ‘:’; px++, i++) (void) putc(*px, fx); for (;i < lmax+P_SPC;i++) (void) putc(‘ ’, fx); /* these count from 1:  * ni[] is currentelement (from 1)  * nc[] is number at start of current line  */ for (px= out[ix]; *px; px++) (void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx);} /*  * put a line of stars (seqs always in out[0], out[1]): dumpblock() */ static stars() stars { int i; register char *p0, *p1, cx, *px; if(!*out[0] || (*out[0] == ‘ ’ && *(p0[0]) == ‘ ’) || !*out[1] || (*out[1]== ‘ ’ && *(po[1]) == ‘ ’)) return; px = star; for (i = lmax+P_SPC; i;i−−) *px++ = ‘ ’; for (p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++){ if (isalpha(*p0) && isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) {cx = ‘*’; nm++; } else if (!dna && _day[*p0− ‘A’][*p1−‘A’] > 0) cx =‘.’; else cx = ‘ ’; } else cx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px =‘\0’; } /*  * strip path or prefix from pn, return len: pr_align()  */static stripname(pn) stripname char *pn; /* file name (may be path) */ {register char *px, *py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’)py = px + 1; if (py) (void) strcpy(pn, py); return(strlen(pn)); } /*  *cleanup() -- cleanup any tmp file  * getseq() -- read in seq, set dna,len, maxlen  * g_calloc() -- calloc() with error checkin  * readjmps()-- get the good jmps, from tmp file if necessary  * writejmps() -- writea filled array of jmps to a tmp file: nw()  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup(); /* cleanup tmp file */ long lseek(); /*  *remove any tmp file if we blow  */ cleanup(i) cleanup int i; { if (fj)(void) unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna,len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upperor lower case  */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file, “r”)) == 0) {fprintf(stderr, “%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr, “%s: malloc() failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ||*line == ‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”, *(py−1))) natgc++; } } *py++ = ‘\0’;*py = ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc() failed %s (n= %d, sz= %d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[] or tmp file, set pp[], reset dmax: main()  */ readjmps() readjmps {int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) { (void)fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr,“%s: can't open() %s\n”, prog, jname); cleanup(1); } } for (i = i0 = i1= 0, dmax0 = dmax, xx = len0; ;i++) { while (1) { for (j =dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmps if(j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset, 0);(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[il] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[il] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; il++; } else if (siz > 0) { /* gapin first seq */ pp[0] .n[i0] = siz; pp[0] .x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw()  */writejmps(ix) writejmps int ix; { char *mktemp(); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp() %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); } 

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3% 

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50% 

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9% 

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides) DNA % nucleic acid sequence identity= (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the PRO-DNA nucleic acid sequence) = 4 dividedby 12 = 33.3%II. Compositions and Methods of the Invention

A. Full-Length PRO Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO polypeptides. In particular, cDNAs encoding various PROpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below. It is noted that proteins produced inseparate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.In particular, Applicants have identified and isolated cDNA encoding aPRO1012 polypeptide, as disclosed in further detail in the Examplesbelow. Using BLAST and FastA seciuence alignment computer programs,Applicants found that various portions of the PRO1012 polypeptide havesequence identity with disulfide isomerase. Accordingly, it is presentlybelieved that PRO1012 polypeptide disclosed in the present applicationis a newly identified member of the ER retained protein family andpossesses activity related to the processing, production and/or foldingof polypeptides typical of the disulfide isomerase family.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

B. PRO Polypeptide Variants

In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence PRO or in various domainsof the PRO described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

PRO polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO polypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res. 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081–1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO

Covalent modifications of PRO are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a PRO polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the PRO. Derivatization with bifunctional agentsis useful, for instance, for crosslinking PRO to a water-insolublesupport matrix or surface for use in the method for purifying anti-PROantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79–86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259–306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem, 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO comprises linking the PROpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The PRO of the present invention may also be modified in a way to form achimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159–2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610–3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204–1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192–194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem. 266:15163–15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393–6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

D. Preparation of PRO

The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

1. Isolation of DNA Encoding PRO

DNA encoding PRO may be obtained from a cDNA library prepared fromtissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to the PRO oroligonucleotides of at least about 20–80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding PRO is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527–537 (1990) and Mansour et al.,Nature, 336:348–352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E.coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO-encodingvectors. Saccharomyces cerevisiae is a commonly used lower eukaryotichost microorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 [1981]; EP 139,383 published May 2, 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968–975 (1991)) such as, e.g., K. lactis (MW98–8C,CBS683, CBS4574; Louvencurt et al., J. Bacteriol., 154(2):737–742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045),K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265–278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259–5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284–289 [1983]; Tilbum et al., Gene,26:205–221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470–1474 [1984]) and A. niger (Kelly Hynes, EMBO J., 4:475–479 [1985]).Methylotropic yeasts are suitable herein and include, but are notlimited to, yeast capable of growth on methanol selected from the generaconsisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,Torulopsis, and Rhodotorula. A list of specific species that areexemplary of this class of yeasts may be found in C. Anthony, TheBiochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. USA, 77:4216(1980)); mouse sertoli cells (T1M4, Mather, Biol.Reprod., 23:243–251 (1980)); human lung cell (W178, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be apart ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, Ipp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21–25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100–270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620–625 (1981); Mantei et al.,Nature, 281:40–46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201–5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of PRO may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

E. Uses for PRO

Nucleotide sequences (or their complement) encoding PRO have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

The full-length native sequence PRO gene, or portions thereof, may beused as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or 35S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO nucleic acids include antisense orsense oligonucleotides comprising a singe-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRODNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of PRO DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 bases in length, about 40 bases in length, about 45bases in length, about 50 bases in length, about 55 bases in length,about 60 bases in length, about 65 bases in length, about 70 bases inlength, about 75 bases in length, about 80 bases in length, about 85bases in length, about 90 bases in length, about 95 bases in length,about 100 bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO coding sequences.

Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

Nucleic acids which encode PRO or its modified forms can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO can be used to construct aPRO “knock out” animal which has a defective or altered gene encodingPRO as a result of homologous recombination between the endogenous geneencoding PRO and altered genomic DNA encoding PRO introduced into anembryonic stem cell of the animal. For example, cDNA encoding PRO can beused to clone genomic DNA encoding PRO in accordance with establishedtechniques. A portion of the genomic DNA encoding PRO can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113–152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the PROpolypeptide.

Nucleic acid encoding the PRO polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143–4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11,205–210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262,4429–4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. U.S. Pat. No. 87,3410–3414(1990). For review of gene markingand gene therapy protocols see Anderson et al., Science 256, 808–813(1992).

The PRO polypeptides described herein may also be employed as molecularweight markers for protein electrophoresis purposes and the isolatednucleic acid sequences may be used for recombinantly expressing thosemarkers.

The nucleic acid molecules encoding the PRO polypeptides or fragmentsthereof described herein are useful for chromosome identification. Inthis regard, there exists an ongoing need to identify new chromosomemarkers, since relatively few chromosome marking reagents, based uponactual sequence data are presently available. Each PRO nucleic acidmolecule of the present invention can be used as a chromosome marker.

The PRO polypeptides and nucleic acid molecules of the present inventionmay also be used diagnostically for tissue typing, wherein the PROpolypeptides of the present invention may be differentially expressed inone tissue as compared to another, preferably in a diseased tissue ascompared to a normal tissue of the same tissue type. PRO nucleic acidmolecules will find use for generating probes for PCR, Northernanalysis, Southern analysis and Western analysis.

The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42–96.

When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a PRO polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795–799 (1996); Yasuda, Biomed. Ther., 27:1221–1223 (1993); Horaet al., Bio/Technology. 8:755–758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdiuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1–41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO polypeptide encoded by the gene identified herein orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the PROpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245–246(1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578–9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789–5793 (1991). Many transcriptional activators, such as yeast GALA,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a PROpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeled PROpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

Another potential PRO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO polypeptide, thereby blocking the normalbiological activity of the PRO polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469–471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

Diagnostic and therapeutic uses of the herein disclosed molecules mayalso be based upon the positive functional assay hits disclosed anddescribed below.

F. Anti-PRO Antibodies

The present invention further provides anti-PRO antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the PRO polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59–103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeuretal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51–63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against PRO.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-PRO antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature 321:522–525(1986); Riechmann etal., Nature, 332:323–329(1988); and Presta, Curr. Op. Struct. Biol.2:593–596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522–525 (1986); Riechmann et al., Nature332:323–327 (1988); Verhoeyen et al., Science, 239:1534–1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86–95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779–783(1992); Lonberg et al., Nature 368 856–859 (1994); Morrison, Nature 368,812–13 (1994); Fishwild et al., Nature Biotechnology 14, 845–51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65–93 (1995).

The antibodies may also be affinity matured using known selection and/ormutagenesis methods as described above. Preferred affinity maturedantibodies have an affinity which is five times, more preferably 10times, even more preferably 20 or 30 times greater than the startingantibody (generally murine, humanized or human) from which the maturedantibody is prepared.

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature 305:537–539 (1983)]. Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published May13, 1993, and in Traunecker et al., EMBO J., 10:3655–3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CHI) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al, Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217–225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547–1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444–6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO polypeptide herein. Alternatively, an anti-PRO polypeptide armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD 16) so as to focus cellular defense mechanisms to thecell expressing the particular PRO polypeptide. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aparticular PRO polypeptide. These antibodies possess a PRO-binding armand an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the PRO polypeptide and further binds tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191–1195(1992) and Shopes, J. Immunol., 148: 2918–2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al CancerResearch, 53: 2560–2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design. 3: 219–230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to acytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286–288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

9. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersin the form of pharmaceutical compositions.

If the PRO polypeptide is intracellular and whole antibodies are used asinhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889–7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

G. Uses for Anti-PRO Antibodies

The anti-PRO antibodies of the invention have various utilities. Forexample, anti-PRO antibodies may be used in diagnostic assays for PRO,e.g., detecting its expression (and in some cases, differentialexpression) in specific cells, tissues, or serum. Various diagnosticassay techniques known in the art may be used, such as competitivebinding assays, director indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147–158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO antibodies also are useful for the affinity purification of PROfrom recombinant cell culture or natural sources. In this process, theantibodies against PRO are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the PROto be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release thePRO from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology 266:460480 (1996)) as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons with a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences usingphrap. In addition, the consensus DNA sequences obtained were often (butnot always) extended using repeated cycles of BLAST or BLAST-2 and phrapto extend the consensus sequence as far as possible using the sources ofEST sequences discussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100–1000 bp in length. The probe sequences are typically 40–55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1–1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278–1280 (1991)) in theunique XhoI and NotI sites.

Example 2 Isolation of cDNA Clones by Amylase Screening

1. Preparation of Oligo dT Primed cDNA Library

mRNA was isolated from a human tissue of interest using reagents andprotocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNAwas used to generate an oligo dT primed cDNA library in the vector pRK5Dusing reagents and protocols from Life Technologies, Gaithersburg, Md.(Super Script Plasmid System). In this procedure, the double strandedcDNA was sized to greater than 1000 bp and the SalI/NotI linkered cDNAwas cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector thathas an sp6 transcription initiation site followed by an SfiI restrictionenzyme site preceding the XhoI/NotI cDNA cloning sites.

2. Preparation of Random Primed cDNA Library

A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500–1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on iceto which was added electrocompetent DH10B bacteria (Life Technologies,20 ml). The bacteria and vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (LifeTechnologies, 1 ml) was added and the mixture was incubated at 37° C.for 30 minutes. The transformants were then plated onto 20 standard 150mm LB plates containing ampicillin and incubated for 16 hours (37° C.).Positive colonies were scraped off the plates and the DNA was isolatedfrom the bacterial pellet using standard protocols, e.g. CsCl-gradient.The purified DNA was then carried on to the yeast protocols below.

The yeast methods were divided into three categories: (1) Transformationof yeast with the plasmid/cDNA combined vector; (2) Detection andisolation of yeast clones secreting amylase; and (3) PCR amplificationof the insert directly from the yeast colony and purification of the DNAfor sequencing and further analysis.

The yeast strain used was HD56–5A (ATCC-90785). This strain has thefollowing genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

Transformation was performed based on the protocol outlined by Gietz etal., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were theninoculated from agar into YEPD complex media broth (100 ml) and grownovernight at 30° C. The YEPD broth was prepared as described in Kaiseret al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., p. 207 (1994). The overnight culture was then diluted toabout 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPD broth (500 ml)and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4–0.5).

The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂₀₀CCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing the prepared cells (100 μl) withfreshly denatured single stranded salmon testes DNA (Lofstrand Labs,Gaithersburg, Md.) and transforming DNA (1 μg, vol.<10 μl) in microfugetubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mMLi₂₀₀CCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5–10 seconds, decanted andresuspended into TE (500 μ1,10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformationwas performed using a single, large scale reaction, wherein reagentamounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lackinguracil (SCD-Ura) prepared as described in Kaiser et al., Methods inYeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p.208–210 (1994). Transformants were grown at 30° C. for 2–3 days.

The detection of colonies secreting amylase was performed by includingred starch in the selective growth media. Starch was coupled to the reddye (Reactive Red-120, Sigma) as per the procedure described by Biely etal., Anal. Biochem., 172:176–179 (1988). The coupled starch wasincorporated into the SCD-Ura agar plates at a final concentration of0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50–100 mM final concentration).

The positive colonies were picked and streaked across fresh selectivemedia (onto 150 mm plates) in order to obtain well isolated andidentifiable single colonies. Well isolated single colonies positive foramylase secretion were detected by direct incorporation of red starchinto buffered SCD-Ura agar. Positive colonies were determined by theirability to break down starch resulting in a clear halo around thepositive colony visualized directly.

4. Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by atoothpick and diluted into sterile water (30 μl) in a 96 well plate. Atthis time, the positive colonies were either frozen and stored forsubsequent analysis or immediately amplified. An aliquot of cells (5 μl)was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide 1 was:

5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′ (SEQ ID NO:553)

The sequence of reverse oligonucleotide 2 was:

5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′ (SEQ ID NO:554)

PCR was then performed as follows:

a. Denature 92° C.,  5 minutes b.  3 cycles of: Denature 92° C., 30seconds Anneal 59° C., 30 seconds Extend 72° C., 60 seconds c.  3 cyclesof: Denature 92° C., 30 seconds Anneal 57° C., 30 seconds Extend 72° C.,60 seconds d. 25 cycles of: Denature 92° C., 30 seconds Anneal 55° C.,30 seconds Extend 72° C., 60 seconds e. Hold  4° C.

The underlined regions of the oligonucleotides annealed to the ADHpromoter region and the amylase region, respectively, and amplified a307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

Following the PCR, an aliquot of the reaction (5 μl) was examined byagarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA(TBE) buffering system as described by Sambrook et al., supra. Clonesresulting in a single strong PCR product larger than 400 bp were furtheranalyzed by DNA sequencing after purification with a 96 Qiaquick PCRclean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis

Various polypeptide-encoding nucleic acid sequences were identified byapplying a proprietary signal sequence finding algorithm developed byGenentech, Inc. (South San Francisco, Calif.) upon ESTs as well asclustered and assembled EST fragments from public (e.g., GenBank) and/orprivate (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, Calif.)databases. The signal sequence algorithm computes a secretion signalscore based on the character of the DNA nucleotides surrounding thefirst and optionally the second methionine codon(s) (ATG) at the 5′-endof the sequence or sequence fragment under consideration. Thenucleotides following the first ATG must code for at least 35unambiguous amino acids without any stop codons. If the first ATG hasthe required amino acids, the second is not examined. If neither meetsthe requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

Example 4 Isolation of cDNA clones Encoding Human PRO Polypeptides

-   -   Using the techniques described in Examples 1 to 3 above,        numerous full-length cDNA clones were identified as encoding PRO        polypeptides as disclosed herein. These cDNAs were then        deposited under the terms of the Budapest Treaty with the        American Type Culture Collection, 10801 University Blvd.,        Manassas, Va. 20110–2209, USA (ATCC) as shown in Table 7 below.

TABLE 7 Material ATCC Dep. No. Deposit Date DNA16438-1387 209771 Apr.14, 1998 DNA19360-2552 203654 Feb. 9, 1999 DNA33455-1548 PTA-127 May 25,1999 DNA37155-2651 PTA-429 Jul. 27, 1999 DNA38269-2654 PTA-432 Jul. 27,1999 DNA40619-1220 209525 Dec. 10, 1997 DNA44174-2513 203577 Jan. 12,1999 DNA44675-2662 PTA-430 Jul. 27, 1999 DNA45408-2615 PTA-203 Jun. 8,1999 DNA48606-1479 203040 Jul. 1, 1998 DNA52753-2656 PTA-611 Aug. 31,1999 DNA53915-1258 209593 Jan. 21, 1998 DNA53991-2553 203649 Feb. 9,1999 DNA54009-2517 203574 Jan. 12, 1999 DNA56055-1643 PTA-129 May 25.1999 DNA57033-1403 209905 May 27, 1998 DNA57252-1453 203585 Jan. 12,1999 DNA58799-1652 203665 Feb. 9, 1999 DNA59770-2652 PTA-427 Jul. 27,1999 DNA59774-2665 PTA-615 Aug. 31, 1999 DNA60281-2518 203582 Jan. 12,1999 DNA60736-2559 203838 Mar. 9, 1999 DNA61875-2653 PTA-428 Jul. 27,1999 DNA62312-2558 203836 Mar. 9, 1999 DNA62849-1604 PTA-205 Jun. 8,1999 DNA66307-2661 PTA-431 Jul. 27, 1999 DNA66677-2535 203659 Feb. 9,1999 DNA71235-1706 203584 Jan. 12, 1999 DNA71289-2547 PTA-126 May 25,1999 DNA73775-1707 PTA-128 May 25, 1999 DNA76385-1692 203664 Feb. 9,1999 DNA76395-2527 203578 Jan. 12, 1999 DNA77622-2516 203554 Dec. 22,1998 DNA77629-2573 203850 Mar. 16, 1999 DNA77645-2648 PTA-45 May 11,1999 DNA79302-2521 203545 Dec. 22, 1998 DNA79865-2519 203544 Dec. 22,1998 DNA80135-2655 PTA-234 Jun. 15, 1999 DNA80794-2568 203848 Mar. 16,1999 DNA80796-2523 203555 Dec. 22, 1998 DNA80840-2605 203949 Apr. 20,1999 DNA80899-2501 203539 Dec. 15, 1998 DNA81228-2580 203871 Mar. 23,1999 DNA81761-2583 203862 Mar. 23, 1999 DNA82358-2738 PTA-510 Aug. 10,1999 DNA82364-2538 203603 Jan. 20, 1999 DNA82424-2566 203813 Mar. 2,1999 DNA82430-2557 203812 Mar. 2, 1999 DNA83500-2506 203391 Oct. 29,1998 DNA83509-2612 203965 Apr. 27, 1999 DNA83560-2569 203816 Mar. 2,1999 DNA84139-2555 203814 Mar. 2, 1999 DNA84141-2556 203810 Mar. 2, 1999DNA84142-2613 PTA-22 May 4, 1999 DNA84318-2520 203580 Jan. 12, 1999DNA84909-2590 203889 Mar. 30, 1999 DNA84912-2610 203964 Apr. 27, 1999DNA84925-2514 203548 Dec. 22, 1998 DNA84928-2564 203817 Mar. 2, 1999DNA84932-2657 PTA-235 Jun. 15, 1999 DNA86592-2607 203968 Apr. 27, 1999DNA86594-2587 203894 Mar. 30, 1999 DNA86647-2591 203893 Mar. 30, 1999DNA87185-2563 203811 Mar. 2, 1999 DNA87656-2582 203867 Mar. 23, 1999DNA87974-2609 203963 Apr. 27, 1999 DNA88001-2565 203815 Mar. 2, 1999DNA88004-2575 203890 Mar. 30, 1999 DNA89220-2608 PTA-130 May 25, 1999DNA89947-2618 203970 Apr. 27, 1999 DNA90842-2574 203845 Mar. 16, 1999DNA91775-2581 203861 Mar. 23, 1999 DNA91779-2571 203844 Mar. 16, 1999DNA92217-2697 PTA-513 Aug. 10, 1999 DNA92219-2541 203663 Feb. 9, 1999DNA92223-2567 203851 Mar. 16, 1999 DNA92225-2603 203950 Apr. 20, 1999DNA92232-2589 203895 Mar. 30, 1999 DNA92233-2599 PTA-134 May 25, 1999DNA92243-2549 203852 Mar. 16, 1999 DNA92253-2671 PTA-258 Jun. 22, 1999DNA92254-2672 PTA-259 Jun. 22, 1999 DNA92255-2584 203866 Mar. 23, 1999DNA92269-2570 203853 Mar. 16, 1999 DNA92288-2588 203892 Mar. 30, 1999DNA92290-2550 203847 Mar. 16, 1999 DNA93012-2622 PTA-21 May 4, 1999DNA93020-2642 PTA-121 May 25, 1999 DNA94830-2604 203951 Apr. 20, 1999DNA94833-2579 203869 Mar. 23, 1999 DNA94838-2658 PTA-232 Jun. 15, 1999DNA94844-2686 PTA-385 Jul. 20, 1999 DNA94854-2586 203864 Mar. 23, 1999DNA96868-2677 PTA-262 Jun. 22, 1999 DNA96871-2683 PTA-381 Jul. 20, 1999DNA96880-2624 PTA-15 May 4, 1999 DNA96986-2660 PTA-239 Jun. 15, 1999DNA96988-2685 PTA-384 Jul. 20, 1999 DNA96995-2709 PTA-475 Aug. 3, 1999DNA97004-2562 203854 Mar. 16, 1999 DNA97005-2687 PTA-378 Jul. 20, 1999DNA97009-2668 PTA-257 Jun. 22, 1999 DNA97013-2667 PTA-231 Jun. 15, 1999DNA98380-2690 PTA-388 Jul. 20, 1999 DNA98561-2696 PTA-620 Aug. 31, 1999DNA98575-2644 PTA-118 May 25, 1999 DNA98593-2694 PTA-477 Aug. 3, 1999DNA98600-2703 PTA-488 Aug. 3, 1999 DNA99391-2572 203849 Mar. 16, 1999DNA99393-2560 203837 Mar. 9, 1999 DNA100276-2684 PTA-380 Jul. 20, 1999DNA100312-2645 PTA-44 May 11, 1999 DNA100902-2646 PTA-42 May 11, 1999DNA102899-2679 PTA-123 May 25, 1999 DNA104875-2720 PTA-482 Aug. 3, 1999DNA105680-2710 PTA-483 Aug. 3, 1999 DNA105779-2708 PTA-485 Aug. 3, 1999DNA105794-2695 PTA-480 Aug. 3, 1999 DNA105838-2702 PTA-476 Aug. 3, 1999DNA107698-2715 PTA-472 Aug. 3, 1999 DNA107701-2711 PTA-487 Aug. 3, 1999DNA107781-2707 PTA-484 Aug. 3, 1999 DNA108670-2744 PTA-546 Aug. 17, 1999DNA108688-2725 PTA-515 Aug. 10, 1999 DNA108769-2765 PTA-861 Oct. 19,1999 DNA108935-2721 PTA-518 Aug. 10, 1999 DNA110700-2716 PTA-512 Aug.10, 1999 DNA111750-2706 PTA-489 Aug. 3, 1999 DNA123430-2755 PTA-614 Aug.31, 1999 DNA125154-2785 PTA-957 Nov. 16,1999 DNA142238-2768 PTA-819 Oct.5, 1999 DNA22779-1130 209280 Sept. 18, 1997 DNA26847-1395 209772 Apr.14, 1998 DNA27864-1155 209375 Oct. 16, 1997 DNA27865-1091 209296 Sept.23, 1997 DNA28497-1130 209279 Sept. 18, 1997 DNA29101-1122 209653 Mar.5, 1998 DNA32286-1191 209385 Oct. 16, 1997 DNA32288-1132 209261 Sept.16, 1997 DNA32290-1164 209384 Oct. 16, 1997 DNA32292-1131 209258 Sept.16, 1997 DNA32298-1132 209257 Sept. 16, 1997 DNA33085-1110 209087 May30, 1997 DNA33087-1158 209381 Oct. 16, 1997 DNA33089-1132 209262 Sept.16, 1997 DNA33092-1202 209420 Oct. 28, 1997 DNA33094-1131 209256 Sept.16, 1997 DNA33107-1135 209251 Sept. 16, 1997 DNA33221-1133 209263 Sept.16, 1997 DNA33223-1136 209264 Sept. 16, 1997 DNA33460-1166 209376 Oct.16, 1997 DNA33473-1176 209391 Oct. 17, 1997 DNA33785-1143 209417 Oct.28, 1997 DNA33786-1132 209253 Sept. 16, 1997 DNA34353-1428 209855 May12, 1998 DNA34392-1170 209526 Dec. 10, 1997 DNA34434-1139 209252 Sept.16, 1997 DNA35558-1167 209374 Oct. 16, 1997 DNA35595-1228 209528 Dec.10, 1997 DNA35638-1216 209265 Sept. 16, 1997 DNA35639-1172 209396 Oct.17, 1997 DNA35663-1129 209201 Aug. 18, 1997 DNA35674-1142 209416 Oct.28, 1997 DNA35841-1173 209403 Oct. 17, 1997 DNA35916-1161 209419 Oct.28, 1997 DNA35918-1174 209402 Oct. 17, 1997 DNA36350-1158 209378 Oct.16, 1997 DNA37140-1234 209489 Nov. 21, 1997 DNA37150-1178 209401 Oct.17, 1997 DNA38260-1180 209397 Oct. 17, 1997 DNA40021-1154 209389 Oct.17, 1997 DNA40587-1231 209438 Nov. 7, 1997 DNA40592-1242 209492 Nov. 21,1997 DNA40620-1183 209388 Oct. 17, 1997 DNA40628-1216 209432 Nov. 7,1997 DNA40981-1234 209439 Nov. 7, 1997 DNA40982-1235 209433 Nov. 7, 1997DNA41234-1242 209618 Feb. 5, 1998 DNA43046-1225 209484 Nov. 21, 1997DNA43316-1237 209487 Nov. 21, 1991 DNA44167-1243 209434 Nov. 7, 1997DNA44184-1319 209704 Mar. 26, 1998 DNA44194-1317 209808 Apr. 28, 1998DNA44196-1353 209847 May 6, 1998 DNA45419-1252 209616 Feb. 5, 1998DNA46777-1253 209619 Feb. 5, 1998 DNA47394-1572 203109 Aug. 11, 1998DNA48331-1329 209715 Mar. 31, 1998 DNA48336-1309 209669 Mar. 11, 1998DNA49142-1430 203002 Jun. 23, 1998 DNA49646-1327 209705 Mar. 26, 1998DNA49821-1562 209981 Jun. 16, 1998 DNA49829-1346 209749 Apr. 7, 1998DNA50921-1458 209859 May 12, 1998 DNA52187-1354 209845 May 6, 1998DNA52196-1348 209748 Apr. 7, 1998 DNA52598-1518 203107 Aug. 11, 1998DNA54228-1366 209801 Apr. 23, 1998 DNA56047-1456 209948 Jun. 9, 1998DNA56112-1379 209883 May 20, 1998 DNA56113-1378 203049 Jul. 1, 1998DNA56352-1358 209846 May 6, 1998 DNA56433-1406 209857 May 12, 1998DNA56439-1376 209864 May 14, 1998 DNA57530-1375 209880 May 20, 1998DNA57689-1385 209869 May 14, 1998 DNA57690-1374 209950 Jun. 9, 1998DNA57693-1424 203008 Jun. 23, 1998 DNA57838-1337 203014 Jun. 23, 1998DNA58721-1475 203110 Aug. 11, 1998 DNA59205-1421 203009 Jun. 23, 1998DNA59215-1425 209961 Jun. 9, 1998 DNA59220-1514 209962 Jun. 9, 1998DNA59294-1381 209866 May 14, 1998 DNA59488-1603 203157 Aug. 25, 1998DNA59588-1571 203106 Aug. 11, 1998 DNA59606-1471 209945 Jun. 9, 1998DNA59620-1463 209989 Jun. 16, 1998 DNA59767-1489 203108 Aug. 11, 1998DNA59777-1480 203111 Aug. 11, 1998 DNA59814-1486 203359 Oct. 20, 1998DNA59839-1461 209988 Jun. 16,1998 DNA59846-1503 209978 Jun. 16, 1998DNA59847-1511 203098 Aug. 4, 1998 DNA60615-1483 209980 Jun. 16, 1998DNA60621-1516 203091 Aug. 4, 1998 DNA60622-1525 203090 Aug. 4, 1998DNA60627-1508 203092 Aug. 4, 1998 DNA60764-1533 203452 Nov. 10, 1998DNA60775-1532 203173 Sept. 1, 1998 DNA61185-1646 203464 Nov. 17, 1998DNA61873-1574 203132 Aug. 18, 1998 DNA62306-1570 203254 Sept. 9, 1998DNA62808-1582 203358 Oct. 20, 1998 DNA62814-1521 203093 Aug. 4, 1998DNA64885-1529 203457 Nov. 3, 1998 DNA64886-1601 203241 Sept. 9, 1998DNA64888-1542 203249 Sept. 9, 1998 DNA64889-1541 203250 Sept. 9, 1998DNA64890-1612 203131 Aug. 18, 1998 DNA64903-1553 203223 Sept. 15, 1998DNA64905-1558 203233 Sept. 15, 1998 DNA65402-1540 203252 Sept. 9, 1998DNA65405-1547 203476 Nov. 17, 1998 DNA65412-1523 203094 Aug. 4, 1998DNA66309-1538 203235 Sept. 15, 1998 DNA66667-1596 203267 Sept. 22, 1998DNA66675-1587 203282 Sept. 22, 1998 DNA68818-2536 203657 Feb. 9, 1999DNA68864-1629 203276 Sept. 22, 1998 DNA68872-1620 203160 Aug. 25, 1998DNA71159-1617 203135 Aug. 18, 1998 DNA73727-1673 203459 Nov. 3, 1998DNA73739-1645 203270 Sept. 22, 1998 DNA76400-2528 203573 Jan. 12, 1999DNA76510-2504 203477 Nov. 17, 1998 DNA76529-1666 203315 Oct. 6, 1998DNA76538-1670 203313 Oct. 6, 1998 DNA77301-1708 203407 Oct. 27, 1998DNA77624-2515 203553 Dec. 22, 1998 DNA79230-2525 203549 Dec. 22, 1998DNA79862-2522 203550 Dec. 22, 1998 DNA80145-2594 PTA-204 Jun. 8, 1999DNA83500-2506 203391 Oct. 29, 1998 DNA84917-2597 203863 Mar. 23, 1999DNA92218-2554 203834 Mar. 9, 1999 DNA96042-2682 PTA-382 Jul. 20, 1999

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations there under (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit and for at least five (5) years afterthe most recent reciuest for the furnishing of a sample of the depositreceived by the depository. The deposits will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures that all restrictionsimposed by the depositor on the availability to the public of thedeposited material will be irrevocably removed upon the granting of thepertinent U.S. patent. assures permanent and unrestricted availabilityof the progeny of the culture of the deposit to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

Example 5 Use of PRO as a Hybridization Probe

The following method describes use of a nucleotide sequence encoding PROas a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO asdisclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO) in humantissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1× SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO can then be identified using standardtechniques known in the art.

Example 6 Expression of PRO in E. coli

This example illustrates preparation of an unglycosylated form of PRO byrecombinant expression in E. coli.

The DNA sequence encoding PRO is initially amplified using selected PCRprimers. The primers should contain restriction enzyme sites whichcorrespond to the restriction enzyme sites on the selected expressionvector. A variety of expression vectors may be employed. An example of asuitable vector is pBR322 (derived from E. coli; see Bolivar et al.,Gene, 2:95 (1977)) which contains genes for ampicillin and tetracyclineresistance. The vector is digested with restriction enzyme anddephosphorylated. The PCR amplified sequences are then ligated into thevector. The vector will preferably include sequences which encode for anantibiotic resistance gene, a trp promoter, a polyhis leader (includingthe first six STII codons, polyhis sequence, and enterokinase cleavagesite), the PRO coding region, lambda transcriptional terminator, and anargu gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO may be expressed in E. coli in a poly-His tagged form, using thefollowing procedure. The DNA encoding PRO is initially amplified usingselected PCR primers. The primers will contain restriction enzyme siteswhich correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq).Transformants are first grown in LB containing 50 mg/mil carbenicillinat 30° C. with shaking until an O.D.600 of 3–5 is reached. Cultures arethen diluted 50–100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate-2H₂O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20–30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6–10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3–5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12–36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2–10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO polypeptide are pooled andthe acetonitrile removed using a gentle stream of nitrogen directed atthe solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14M sodium chloride and 4% mannitol by dialysis or by gel filtration usingG25 Superfine (Pharmacia) resins equilibrated in the formulation bufferand sterile filtered.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 7 Expression of PRO in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof PRO by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the PRO DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-PRO.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRODNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappayaet al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl,0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added, dropwise, 500 μlof 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitateis allowed to form for 10 minutes at 25° C. The precipitate is suspendedand added to the 293 cells and allowed to settle for about four hours at37° C. The culture medium is aspirated off and 2 ml of 20% glycerol inPBS is added for 30 seconds. The 293 cells are then washed with serumfree medium, fresh medium is added and the cells are incubated for about5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

In another embodiment, PRO can be expressed in CHO cells. The pRK5-PROcan be transfected into CHO cells using known reagents such as CaPO₄ orDEAE-dextran. As described above, the cell cultures can be incubated,and the medium replaced with culture medium (alone) or medium containinga radiolabel such as ³⁵S-methionine. After determining the presence ofPRO polypeptide, the culture medium may be replaced with serum freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested. The medium containing theexpressed PRO can then be concentrated and purified by any selectedmethod.

Epitope-tagged PRO may also be expressed in host CHO cells. The PRO maybe subcloned out of the pRK5 vector. The subclone insert can undergo PCRto fuse in frame with a selected epitope tag such as a poly-his tag intoa Baculovirus expression vector. The poly-his tagged PRO insert can thenbe subcloned into a SV40 driven vector containing a selection markersuch as DHFR for selection of stable clones. Finally, the CHO cells canbe transfected (as described above) with the SV40 driven vector.Labeling may be performed, as described above, to verify expression. Theculture medium containing the expressed poly-His tagged PRO can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774–1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1–2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2–3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4–5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation. Many of the PRO polypeptidesdisclosed herein were successfully expressed as described above.

Example 8 Expression of PRO in Yeast

The following method describes recombinant expression of PRO in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 9 Expression of PRO in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of PRO inBaculovirus-infected insect cells.

The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pV_(L)1393 (Novagen). Briefly,the sequence encoding PRO or the desired portion of the coding sequenceof PRO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4–5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175–179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged PRO are pooled and dialyzedagainst loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 10 Preparation of Antibodies that Bind PRO

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra Immunogens that may beemployed include purified PRO, fusion proteins containing PRO, and cellsexpressing recombinant PRO on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the PRO immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1–100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO. Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against PRO is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 11 Purification of PRO Polypeptides Using Specific Antibodies

Native or recombinant PRO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO polypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-PRO polypeptide antibody toan activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PROpolypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2–3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 12 Drug Screening

This invention is particularly useful for screening compounds by usingPRO polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO polypeptide-associated diseaseor disorder. These methods comprise contacting such an agent with an PROpolypeptide or fragment thereof and assaying (I) for the presence of acomplex between the agent and the PRO polypeptide or fragment, or (ii)for the presence of a complex between the PRO polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO polypeptide or fragment is typically labeled.After suitable incubation, free PRO polypeptide or fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind to PROpolypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO polypeptide, the peptide test compounds arereacted with PRO polypeptide and washed. Bound PRO polypeptide isdetected by methods well known in the art. Purified PRO polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. In addition, non-neutralizing antibodies can beused to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 13 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (c.f., Hodgson, Bio/Technology, 9: 19–21 (1991)).

In one approach, the three-dimensional structure of the PRO polypeptide,or of an PRO polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry. 31:7796–7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742–746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

Example 14 Identification of PRO Polypeptides That Stimulate TNF-αRelease In Human Blood (Assay 128)

This assay shows that certain PRO polypeptides of the present inventionact to stimulate the release of TNF-α in human blood. PRO polypeptidestesting positive in this assay are useful for, among other things,research purposes where stimulation of the release of TNF-α would bedesired and for the therapeutic treatment of conditions wherein enhancedTNF-α release would be beneficial. Specifically, 200 μl of human bloodsupplemented with 50 mM Hepes buffer (pH 7.2) is aliquoted per well in a96 well test plate. To each well is then added 300 μl of either the testPRO polypeptide in 50 mM Hepes buffer (at various concentrations) or 50mM Hepes buffer alone (negative control) and the plates are incubated at37° C. for 6 hours. The samples are then centrifuged and 50 μl of plasmais collected from each well and tested for the presence of TNF-α byELISA assay. A positive in the assay is a higher amount of TNF-α in thePRO polypeptide treated samples as compared to the negative controlsamples.

The following PRO polypeptides tested positive in this assay: PRO195,PRO202, PRO215, PRO221, PRO217, PRO222, PRO198, PRO245, PRO172, PRO265,PRO266, PRO344, PRO337, PRO322, PRO1286, PRO1279, PRO1338 and PRO1343.

Example 15 Detection of Polypeptides That Affect Glucose or FFA Uptakein Skeletal Muscle (Assay 106)

This assay is designed to determine whether PRO polypeptides show theability to affect glucose or FFA uptake by skeletal muscle cells. PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of disorders where either thestimulation or inhibition of glucose uptake by skeletal muscle would bebeneficial including, for example, diabetes or hyper- orhypo-insulinemia.

In a 96 well format, PRO polypeptides to be assayed are added to primaryrat differentiated skeletal muscle, and allowed to incubate overnight.Then fresh media with the PRO polypeptide and +/− insulin are added tothe wells. The sample media is then monitored to determine glucose andFFA uptake by the skeletal muscle cells. The insulin will stimulateglucose and FFA uptake by the skeletal muscle, and insulin in mediawithout the PRO polypeptide is used as a positive control, and a limitfor scoring. As the PRO polypeptide being tested may either stimulate orinhibit glucose and FFA uptake, results are scored as positive in theassay if greater than 1.5 times or less than 0.5 times the insulincontrol.

The following PRO polypeptides tested positive as being capable ofaffecting glucose and/or FFA uptake by skeletal muscle in this assay:PRO182, PRO366, PRO198, PRO172 and PRO719.

Example 16 Chondrocyte Re-differentiation Assay (Assay 110)

This assay shows that certain polypeptides of the invention act toinduce redifferentiation of chondrocytes, therefore, are expected to beuseful for the treatment of various bone and/or cartilage disorders suchas, for example, sports injuries and arthritis. The assay is performedas follows. Porcine chondrocytes are isolated by overnight collagenasedigestion of articulary cartilage of metacarpophalangeal joints of 4–6month old female pigs. The isolated cells are then seeded at 25,000cells/cm² in Ham F-12 containing 10% FBS and 4 μg/ml gentamycin. Theculture media is changed every third day and the cells are then seededin 96 well plates at 5,000 cells/well in 100 μl of the same mediawithout serum and 100 μl of the test PRO polypeptide, 5 nM staurosporin(positive control) or medium alone (negative control) is added to give afinal volume of 200 μl/well. After 5 days of incubation at 37° C., apicture of each well is taken and the differentiation state of thechondrocytes is determined. A positive result in the assay occurs whenthe redifferentiation of the chondrocytes is determined to be moresimilar to the positive control than the negative control.

The following polypeptide tested positive in this assay: PRO182, PRO366,PRO198 and PRO1868.

Example 17 Chondrocyte Proliferation Assay (Assay 111)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to induce the proliferation and/orredifferentiation of chondrocytes in culture. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of various bone and/or cartilage disorders suchas, for example, sports injuries and arthritis.

Porcine chondrocytes are isolated by overnight collagenase digestion ofarticular cartilage of the metacarpophalangeal joint of 4–6 month oldfemale pigs. The isolated cells are then seeded at 25,000 cells/cm² inHam F-12 containing 10% FBS and 4 μg/ml gentamycin. The culture media ischanged every third day and the cells are reseeded to 25,000 cells/cm²every five days. On day 12, the cells are seeded in 96 well plates at5,000 cells/well in 100%1 of the same media without serum and 100 μL ofeither serum-free medium (negative control), staurosporin (finalconcentration of 5 nM; positive control) or the test PRO polypeptide areadded to give a final volume of 200 μl/well. After 5 days at 37° C., 20μl of Alamar blue is added to each well and the plates are incubated foran additional 3 hours at 37° C. The fluorescence is then measured ineach well (Ex:530 nm; Em: 590 nm). The fluorescence of a platecontaining 200 μl of the serum-free medium is measured to obtain thebackground. A positive result in the assay is obtained when thefluorescence of the PRO polypeptide treated sample is more like that ofthe positive control than the negative control.

The following PRO polypeptides tested positive in this assay: PRO202,PRO224, PRO172 and PRO1312.

Example 18 Detection of PRO Polypeptides That Affect Glucose or FFAUptake by Primary Rat Adipocytes (Assay 94)

This assay is designed to determine whether PRO polypeptides show theability to affect glucose or FFA uptake by adipocyte cells. PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of disorders where either thestimulation or inhibition of glucose uptake by adipocytes would bebeneficial including, for example, obesity, diabetes or hyper- orhypo-insulinemia.

In a 96 well format, PRO polypeptides to be assayed are added to primaryrat adipocytes, and allowed to incubate overnight. Samples are taken at4 and 16 hours and assayed for glycerol, glucose and FFA uptake. Afterthe 16 hour incubation, insulin is added to the media and allowed toincubate for 4 hours. At this time, a sample is taken and glycerol,glucose and FFA uptake is measured. Media containing insulin without thePRO polypeptide is used as a positive reference control. As the PROpolypeptide being tested may either stimulate or inhibit glucose and FFAuptake, results are scored as positive in the assay if greater than 1.5times or less than 0.5 times the insulin control.

The following PRO polypeptides tested positive as being capable ofaffecting glucose and/or FFA uptake in this assay: PRO202, PRO211,PRO344 and PRO1338.

Example 19 Gene Expression in Bovine Pericytes (Assay 105)

This assay is designed to identify PRO polypeptides which activate geneexpression in pericytes. Such polypeptides would be expected to beuseful as growth factors and/or for situations where the activation ofgene expression is desired or beneficial. Bovine pericytes are plated on60 mm culture dishes in growth media for 1 week. On day 1, various PROpolypeptides are diluted (1%) and incubated with the pericytes for 1,4and 24 hr. timepoints. The cells are harvested and the RNA isolatedusing TR1-Reagent following the included instructions. The RNA is thenquantified by reading the 260/280 OD using a spectrophotometer. The geneexpression analysis is done by TaqMan reactions using Perkin Elmerreagents and specially designed bovine probes and primers. Expression ofthe following genes is analyzed: GAPDH, beta-integrin, connective tissuegrowth factor (CTGF), ICAM-1, monocyte chemoattractantprotein-1 (MCP-1),osteopontin, transforming growth factor-beta (TGF-beta), TGF-betareceptor, tissue inhibitor of metalloproteinase (TIMP), tissue factor(TF), VEGF-A, thrombospondin, VEGF-β, angiopoeitin-2, and collagenase.Replicates are then averaged and the SD determined. The gene expressionlevels are then normalized to GAPDH. These are then normalized to theexpression levels obtained with a protein (PIN32) which does notsignificantly induce gene expression in bovine pericytes when comparedto untreated controls. Any PRO polypeptide that gives a gene expressionlevel 2-fold or higher over the PIN32 control is considered a positivehit.

The following PRO polypeptides tested positive in this assay: PRO366.

Example 20 Identification of PRO Polypeptides That Activate Pericytes(Assay 125)

This assay shows that certain polypeptides of the invention act toactivate proliferation of pericyte cells and, therefore, are useful notonly as diagnostic markers for particular types of pericyte-associatedtumors but also for giving rise to antagonists which would be expectedto be useful for the therapeutic treatment of pericyte-associatedtumors. Such PRO polypeptides also would be expected to be useful asgrowth factors and/or for situations where the induction of cellproliferation is desired or beneficial. Activation of pericyteproliferation also correlates with the induction of angiogenesis and, assuch, PRO polypeptides capable of inducing pericyte proliferation wouldbe expected to be useful for the treatment of conditions where inducedangiogenesis would be beneficial including, for example, wound healing,and the like. Specifically, on day 1, pericytes are received from VECTechnologies, and all but 5 ml media is removed from the flask. On day2, the pericytes are trypsinized, washed, spun and plated on 96 wellplates. On day 7, the media is removed and the pericytes are treatedwith 100 μl of either the specific PRO polypeptide or control treatments(positive control =DME+5% +/−PDGF @ 500 ng/μl; negative control=PIN32, apolypeptide determined to have no significant effect on pericyteproliferation). C-fos and GAPDH gene expression levels are thendetermined and the replicates are averaged and the SD is determined. Thec-fos values are normalized to GAPDH and the results are expressed asfold increase over PIN32. Anything providing at least a 2-fold or higherresponse as compared to the negative control is considered positive forthe assay.

The following polypeptides tested positive in this assay: PRO366.

Example 21 Ability of PRO Polypeptides to Stimulate the Release ofProteoglycans from Cartilage (Assay 97)

The ability of various PRO polypeptides to stimulate the release ofproteoglycans from cartilage tissue was tested as follows.

The metacarphophalangeal joint of 4–6 month old pigs was asepticallydissected, and articular cartilage was removed by free hand slicingbeing careful to avoid the underlying bone. The cartilage was minced andcultured in bulk for 24 hours in a humidified atmosphere of 95% air, 5%CO₂ in serum free (SF) media (DME/F12 1:1) with 0.1% BSA and 100 U/mlpenicillin and 100 μg/ml streptomycin. After washing three times,approximately 100 mg of articular cartilage was aliquoted into micronicstubes and incubated for an additional 24 hours in the above SF media.PRO polypeptides were then added at 1% either alone or in combinationwith 18 ng/ml interleukin-la, a known stimulator of proteoglycan releasefrom cartilage tissue. The supernatant was then harvested and assayedfor the amount of proteoglycans using the 1,9-dimethyl-methylene blue(DMB) colorimetric assay (Farndale and Buttle, Biochem. Biophys. Acta883:173–177 (1985)). A positive result in this assay indicates that thetest polypeptide will find use, for example, in the treatment ofsports-related joint problems, articular cartilage defects,osteoarthritis or rheumatoid arthritis.

When various PRO polypeptides were tested in the above assay, thepolypeptides demonstrated a marked ability to stimulate release ofproteoglycans from cartilage tissue both basally and after stimulationwith interleukin-1α and at 24 and 72 hours after treatment, therebyindicating that these PRO polypeptides are useful for stimulatingproteoglycan release from cartilage tissue. As such, these PROpolypeptides are useful for the treatment of sports-related jointproblems, articular cartilage defects, osteoarthritis or rheumatoidarthritis. The polypeptides testing positive in this assay are: PRO216.

Example 22 Proliferation of Rat Utricular Supporting Cells (Assay 54)

This assay shows that certain polypeptides of the invention act aspotent mitogens for inner ear supporting cells which are auditory haircell progenitors and, therefore, are useful for inducing theregeneration of auditory hair cells and treating hearing loss inmammals. The assay is performed as follows. Rat UEC-4 utricularepithelial cells are aliquoted into 96 well plates with a density of3000 cells/well in 200 μl of serum-containing medium at 33° C. The cellsare cultured overnight and are then switched to serum-free medium at 37°C. Various dilutions of PRO polypeptides (or nothing for a control) arethen added to the cultures and the cells are incubated for 24 hours.After the 24 hour incubation, ³H-thymidine (1 μCi/well) is added and thecells are then cultured for an additional 24 hours. The cultures arethen washed to remove unincorporated radiolabel, the cells harvested andCpm per well determined. Cpm of at least 30% or greater in the PROpolypeptide treated cultures as compared to the control cultures isconsidered a positive in the assay.

The following polypeptides tested positive in this assay: PRO172.

Example 23 Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay(Assay 24)

This example shows that certain polypeptides of the invention are activeas a stimulator of the proliferation of stimulated T-lymphocytes.Compounds which stimulate proliferation of lymphocytes are usefultherapeutically where enhancement of an immune response is beneficial. Atherapeutic agent may take the form of antagonists of the polypeptide ofthe invention, for example, murine-human chimeric, humanized or humanantibodies against the polypeptide.

The basic protocol for this assay is described in Current Protocols inImmunology, unit 3.12; edited by J E Coligan, A M Kruisbeek, D HMarglies, E M Shevach, W Strober, National Institutes of Health,Published by John Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatorPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO₂) and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate). The stimulator PBMCs areprepared by irradiating the cells (about 3000 Rads).

The assay is prepared by plating in triplicate wells a mixture of:

-   -   100:1 of test sample diluted to 1% or to 0.1%,    -   50:1 of irradiated stimulator cells, and    -   50:1 of responder PBMC cells.        100 microliters of cell culture media or 100 microliter of        CD4-IgG is used as the control. The wells are then incubated at        37° C., 5% CO₂ for 4 days. On day 5, each well is pulsed with        tritiated thymidine (1.0 mC/well; Amersham). After 6 hours the        cells are washed 3 times and then the uptake of the label is        evaluated.

In another variant of this assay, PBMCs are isolated from the spleens ofBalb/c mice and C57B6 mice. The cells are teased from freshly harvestedspleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

Positive increases over control are considered positive with increasesof greater than or equal to 180% being preferred. However, any valuegreater than control indicates a stimulatory effect for the testprotein.

The following PRO polypeptides tested positive in this assay: PRO344.

Example 24 Pericyte c-Fos Induction (Assay 93)

This assay shows that certain polypeptides of the invention act toinduce the expression of c-fos in pericyte cells and, therefore, areuseful not only as diagnostic markers for particular types ofpericyte-associated tumors but also for giving rise to antagonists whichwould be expected to be useful for the therapeutic treatment ofpericyte-associated tumors. Induction of c-fos expression in pericytesis also indicative of the induction of angiogenesis and, as such, PROpolypeptides capable of inducing the expression of c-fos would beexpected to be useful for the treatment of conditions where inducedangiogenesis would be beneficial including, for example, wound healing,and the like. Specifically, on day 1, pericytes are received from VECTechnologies and all but 5 ml of media is removed from flask. On day 2,the pericytes are trypsinized, washed, spun and then plated onto 96 wellplates. On day 7, the media is removed and the pericytes are treatedwith 1100 μl of PRO polypeptide test samples and controls (positivecontrol=DME+5% serum+/−PDGF at 500 ng/ml; negative control=protein 32).Replicates are averaged and SD/CV are determined. Fold increase overProtein 32 (buffer control) value indicated by chemiluminescence units(RLU) luminometer reading verses frequency is plotted on a histogram.Two-fold above Protein 32 value is considered positive for the assay.ASY Matrix: Growth media=low glucose DMEM=20% FBS+1× pen strep+1×fungizone. Assay Media=low glucose DMEM+5% FBS. The followingpolypeptides tested positive in this assay: PRO301, PRO619, PRO1066 andPRO1265.

Example 25 Cytokine Release Assay (Assay 120)

This assay is designed to determine whether PRO polypeptides of thepresent invention are capable of inducing the release of cytokines fromperipheral blood mononuclear cells (PBMCs). PRO polypeptides capable ofinducing the release of cytokines from PBMCs are useful from thetreatment of conditions which would benefit from enhanced cytokinerelease and will be readily evident to those of ordinary skill in theart. Specifically, 1×10⁶ cells/ml of peripheral blood mononuclear cells(PBMC) are cultured with 1% of a PRO polypeptide for 3 days in completeRPMI media. The supernatant is then harvested and tested for increasedconcentrations of various cytokines by ELISA as compared to a human IgGtreated control. A positive in the assay is a 10-fold or greaterincrease in cytokine concentration in the PRO polypeptide treated sampleas compared to the human IgG treated control.

The following polypeptides tested positive in this assay: PRO526 andPRO1343.

Example 26 Inhibition of A-Peptide Binding to Factor VIIA (Assay 118)

This assay is designed to identify PRO polypeptides which are capable ofinhibiting the binding of A-peptide to factor VIIA, thereby affectingthe blood coagulation cascade. PRO polypeptides testing positive in thisassay are expected to be useful for the treatment of conditions wherealteration of the blood coagulation cascade would be beneficialincluding, for example, stroke, heart attack and various coagulationdisorders. These PRO polypeptides are also useful for the identificationof agonist and antagonist molecules which would also be useful fortreatment of those conditions.

Specifically, 384 well plates are coated with soluble factor VIIA andare incubated overnight at 4° C. The wells are then decanted and areblocked by the addition of 0.5% BSA for 1 hour. The wells are thenwashed and 20 μl of biotinylated A-peptide and either variousconcentration of the PRO polypeptide (test) or nothing (negativecontrol) are added to each well. The plates are then incubated for 1hour at room temperature. The wells are again washed and then 40 μl ofstreptavidin-europium is added to each well. The plates are thenincubated for 30 minutes at room temperature and then washed. 40 μl of afluorescence enhancement solution is then added to each well, the platesincubated for 5 minutes at room temperature and each well is then readon Wallac Victor reader under europium delayed fluorescence settings.Percent inhibition of binding of the A-peptide to the factor VIIA isthen determined (as compared to the negative control), wherein apositive in the assay is a percent inhibition of 30% or greater.

The following PRO polypeptides tested positive in this assay: PRO182.

Example 27 Inhibition of Adipocyte Differentiation Assay (Assay 66)

This assay is designed to identify PRO polypeptides which are capable ofinhibiting insulin-induced differentiation of adipocytes. PROpolypeptides testing positive in this assay would be expected to beuseful for the treatment of conditions associated with obesity,diabetes, etc.

Specifically, 3T3-L1 cells are seeded into the wells of 96 well platesat 6×10⁴ cells/well and allowed to grow to confluency for 7 days. At day7, the cells are treated with various concentrations of the PROpolypeptide (or nothing for the negative control) in the presence of 1g/mil insulin, 0.25×10⁻⁶ M dexamethasone and 0.5 mM IBMX. The samplesare then incubated at 37° C. in 7% CO₂ for 2 days. After the incubation,the media is removed by aspiration and the cells are washed with PBS andre-exposed to the PRO polypeptide (or nothing for the negative control)and 1 μg/ml insulin. After 5 days, the media is removed and replacedwith fresh PRO polypeptide (or nothing for the negative control) andinsulin. After 5 days, the cells are lysed and the cell lysate isassayed using Sigma's Triglyceride [α]kit (Sigma procedure #336). Apositive in the assay is 20% greater inhibition of adipocytedifferentiation in the PRO polypeptide treated samples as compared tothe negative control.

The following PRO polypeptides tested positive in this assay: PRO185 andPRO198.

Example 28 HUVEC Stimulation by PRO Polypeptides (Assay 131)

This assay is designed to identify PRO polypeptides which are capable ofstimulating the proliferation of HUVEC cells. PRO polypeptides testingpositive in this assay would be expected to be useful for inducingangiogenesis for the treatment of conditions where angiogenesis would bebeneficial including, for example, wound healing, and the like.Antagonists of these PRO polypeptides would be expected to be useful forinhibiting angiogenesis for the treatment of, for example, tumors, andthe like.

Specifically, COSTAR® flat bottom black plates are treated withfibronectin for 20 minutes and then washed twice with PBS. HUVEC cellsare then plated at 2000 cells/well in an appropriate growth medium. Theplates are then incubated overnight and then the PRO polypeptide (1%final concentration), nothing (negative control) or IL1β (3.3 ng/mlfinal concentration; positive control) is added. The plates are againincubated overnight, stained with ICAM1-Cy5 and read on FMAT. A positivein the assay is a 2-fold or greater increase in fluorescence as comparedto the positive control.

The following PRO polypeptides tested positive in this assay: PRO222.

Example 29 Promotion of Chondrocyte Redifferentiation (Assay 129)

This assay is designed to determine whether PRO polypeptides of thepresent invention show the ability to induce the proliferation and/orredifferentiation of chondrocytes in culture. PRO polypeptides testingpositive in this assay would be expected to be useful for thetherapeutic treatment of various bone and/or cartilage disorders suchas, for example, sports injuries and arthritis.

Porcine chondrocytes are isolated by overnight collagenase digestion ofarticular cartilage of the metacarpophalangeal joint of 4–6 month oldfemale pigs. The isolated cells are then seeded at 25,000 cells/cm² inHam F-12 containing 10% FBS and 4 μg/ml gentamycin. The culture media ischanged every third day. On day 12, the cells are seeded in 96 wellplates at 5,000 cells/well in 100 μl of the same media without serum and100 μl of either serum-free medium (negative control), staurosporin(final concentration of 5 nM; positive control) or the test PROpolypeptide are added to give a final volume of 200 μl/well. After 5days at 37° C., 22 μl of media containing 100 μg/ml Hoechst 33342 and 50μg/ml 5-CFDA is added to each well and incubated for an additional 10minutes at 37° C. A picture of the green fluorescence is taken for eachwell and the differentiation state of the chondrocytes is calculated bymorphometric analysis. A positive result in the assay is obtained whenthe >50% of the PRO polypeptide treated cells are differentiated(compared to the background obtained by the negative control).

The following PRO polypeptides tested positive in this assay: PRO301.

Example 30 Microarray Analysis to Detect Overexpression of PROPolypeptides in Cancerous Tumors

Nucleic acid microarrays, often containing thousands of gene sequences,are useful for identifying differentially expressed genes in diseasedtissues as compared to their normal counterparts. Using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. ThecDNA probes are then hybridized to an array of nucleic acids immobilizedon a solid support. The array is configured such that the sequence andposition of each member of the array is known. For example, a selectionof genes known to be expressed in certain disease states may be arrayedon a solid support. Hybridization of a labeled probe with a particulararray member indicates that the sample from which the probe was derivedexpresses that gene. If the hybridization signal of a probe from a test(disease tissue) sample is greater than hybridization signal of a probefrom a control (normal tissue) sample, the gene or genes overexpressedin the disease tissue are identified. The implication of this result isthat an overexpressed protein in a diseased tissue is useful not only asa diagnostic marker for the presence of the disease condition, but alsoas a therapeutic target for treatment of the disease condition.

The methodology of hybridization of nucleic acids and microarraytechnology is well known in the art. In the present example, thespecific preparation of nucleic acids for hybridization and probes,slides, and hybridization conditions are all detailed in U.S.Provisional Patent Application Serial No. 60/193,767, filed on Mar. 31,2000 and which is herein incorporated by reference.

In the present example, cancerous tumors derived from various humantissues were studied for PRO polypeptide-encoding gene expressionrelative to non-cancerous human tissue in an attempt to identify thosePRO polypeptides which are overexpressed in cancerous tumors. Two setsof experimental data were generated. In one set, cancerous human colontumor tissue and matched non-cancerous human colon tumor tissue from thesame patient (“matched colon control”) were obtained and analyzed forPRO polypeptide expression using the above described microarraytechnology. In the second set of data, cancerous human tumor tissue fromany of a variety of different human tumors was obtained and compared toa “universal” epithelial control sample which was prepared by poolingnon-cancerous human tissues of epithelial origin, including liver,kidney, and lung. mRNA isolated from the pooled tissues represents amixture of expressed gene products from these different tissues.Microarray hybridization experiments using the pooled control samplesgenerated a linear plot in a 2-color analysis. The slope of the linegenerated in a 2-color analysis was then used to normalize the ratios of(test:control detection) within each experiment. The normalized ratiosfrom various experiments were then compared and used to identifyclustering of gene expression. Thus, the pooled “universal control”sample not only allowed effective relative gene expressiondeterminations in a simple 2-sample comparison, it also allowedmulti-sample comparisons across several experiments.

In the present experiments, nucleic acid probes derived from the hereindescribed PRO polypeptide-encoding nucleic acid sequences were used inthe creation of the microarray and RNA from the tumor tissues listedabove were used for the hybridization thereto. A value based upon thenormalized ratio:experimental ratio was designated as a “cutoff ratio”.Only values that were above this cutoff ratio were determined to besignificant. Table 8 below shows the results of these experiments,demonstrating that various PRO polypeptides of the preent invention aresignificantly overexpressed in various human tumor tissues as comparedto a non-cancerous human tissue control. As described above, these datademonstrate that the PRO polypeptides of the present invention areuseful not only as diagnostic markers for the presence of one or morecancerous tumors, but also serve as therapeutic targets for thetreatment of those tumors.

TABLE 8 Molecule is overexpressed in: as compared to: PRO177 breasttumor universal normal control PRO177 liver tumor universal normalcontrol PRO177 lung tumor universal normal control PRO3574 breast tumoruniversal normal control PRO3574 colon tumor matched normal coloncontrol PRO1280 breast tumor universal normal control PRO1280 lung tumoruniversal normal control PRO4984 lung tumor universal normal controlPRO4988 colon tumor universal normal control PRO4988 lung tumoruniversal normal control PRO305 lung tumor universal normal controlPRO305 colon tumor universal normal control PRO1866 prostate tumoruniversal normal control PRO1866 lung tumor universal normal controlPRO1866 colon tumor universal normal control PRO4996 breast tumoruniversal normal control PRO4996 lung tumor universal normal controlPRO4406 lung tumor universal normal control PRO4406 colon tumoruniversal normal control PRO1120 colon tumor universal normal controlPRO1120 breast tumor universal normal control PRO1120 rectal tumoruniversal normal control PRO4990 lung tumor universal normal controlPRO738 cervical tumor universal normal control PRO738 lung tumoruniversal normal control PRO738 breast tumor universal normal controlPRO3577 lung tumor universal normal control PRO1879 breast tumoruniversal normal control PRO1879 lung tumor universal normal controlPRO1879 colon tumor universal normal control PRO1471 lung tumoruniversal normal control PRO1076 prostate tumor universal normal controlPRO1483 lung tumor universal normal control PRO4985 rectal tumoruniversal normal control PRO4985 colon tumor universal normal controlPRO4985 breast tumor universal normal control PRO4985 lung tumoruniversal normal control PRO5000 lung tumor universal normal controlPRO1881 liver tumor universal normal control PRO1881 lung tumoruniversal normal control PRO1881 breast tumor universal normal controlPRO4314 lung tumor universal normal control PRO4314 breast tumoruniversal normal control PRO4987 lung tumor universal normal controlPRO4313 lung tumor universal normal control PRO4313 breast tumoruniversal normal control PRO4799 colon tumor universal normal controlPRO4995 liver tumor universal normal control PRO4995 colon tumoruniversal normal control PRO4995 colon tumor matched normal coloncontrol PRO1341 prostate tumor universal normal control PRO1341 lungtumor universal normal control PRO1341 colon tumor universal normalcontrol PRO1341 colon tumor matched normal colon control PRO1777 lungtumor universal normal control PRO1777 colon tumor matched normal coloncontrol PRO3580 lung tumor universal normal control PRO3580 prostatetumor universal normal control PRO1779 lung tumor universal normalcontrol PRO1779 colon tumor universal normal control PRO1779 cervicaltumor universal normal control PRO1754 breast tumor universal normalcontrol PRO1754 lung tumor universal normal control PRO1906 breast tumoruniversal normal control PRO1906 colon tumor universal normal controlPRO1906 prostate tumor universal normal control PRO1870 breast tumoruniversal normal control PRO4329 lung tumor universal normal controlPRO4979 colon tumor universal normal control PRO1885 rectal tumoruniversal normal control PRO1885 colon tumor universal normal controlPRO1885 colon tumor matched normal colon control PRO1882 prostate tumoruniversal normal control PRO1882 lung tumor universal normal controlPRO1882 colon tumor universal normal control PRO1882 breast tumoruniversal normal control PRO1882 cervical tumor universal normal controlPRO4989 rectal tumor universal normal control PRO4989 breast tumoruniversal normal control PRO4989 colon tumor matched normal coloncontrol PRO4989 colon tumor universal normal control PRO4323 lung tumoruniversal normal control PRO4323 liver tumor universal normal controlPRO1886 breast tumor universal normal control PRO1886 lung tumoruniversal normal control PRO1886 rectal tumor universal normal controlPRO4395 colon tumor universal normal control PRO4395 prostate tumoruniversal normal control PRO4395 lung tumor universal normal controlPRO4395 cervical tumor universal normal control PRO1782 colon tumoruniversal normal control PRO1782 lung tumor universal normal controlPRO4388 lung tumor universal normal control PRO4341 breast tumoruniversal normal control PRO4341 lung tumor universal normal controlPRO3438 lung tumor universal normal control PRO4321 breast tumoruniversal normal control PRO4321 lung tumor universal normal controlPRO4321 colon tumor universal normal control PRO4304 breast tumoruniversal normal control PRO4304 lung tumor universal normal controlPRO4403 colon tumor universal normal control PRO4403 breast tumoruniversal normal control PRO4403 lung tumor universal normal controlPRO4324 lung tumor universal normal control PRO4324 breast tumoruniversal normal control PRO4303 cervical tumor universal normal controlPRO4303 lung tumor universal normal control PRO4303 breast tumoruniversal normal control PRO4303 colon tumor universal normal controlPRO4303 prostate tumor universal normal control PRO4305 breast tumoruniversal normal control PRO4305 lung tumor universal normal controlPRO4305 colon tumor universal normal control PRO4305 liver tumoruniversal normal control PRO4404 lung tumor universal normal controlPRO4404 breast tumor universal normal control PRO4404 rectal tumoruniversal normal control PRO1884 lung tumor universal normal controlPRO4349 colon tumor universal normal control PRO4349 lung tumoruniversal normal control PRO4401 colon tumor universal normal controlPRO4401 lung tumor universal normal control PRO1867 lung tumor universalnormal control PRO1867 liver tumor universal normal control PRO4319breast tumor universal normal control PRO4319 lung tumor universalnormal control PRO4991 lung tumor universal normal control PRO4991 colontumor universal normal control PRO4398 lung tumor universal normalcontrol PRO4346 lung tumor universal normal control PRO4350 colon tumoruniversal normal control PRO4350 prostate tumor universal normal controlPRO4350 lung tumor universal normal control PRO4318 prostate tumoruniversal normal control PRO4318 lung tumor universal normal controlPRO4340 breast tumor universal normal control PRO4340 lung tumoruniversal normal control PRO4400 breast tumor universal normal controlPRO4400 lung tumor universal normal control PRO4320 lung tumor universalnormal control PRO4409 lung tumor universal normal control PRO4409cervical tumor universal normal control PRO4409 colon tumor universalnormal control PRO4399 lung tumor universal normal control PRO4399breast tumor universal normal control PRO4418 lung tumor universalnormal control PRO4418 breast tumor universal normal control PRO4330cervical tumor universal normal control PRO4330 colon tumor matchednormal colon control PRO4339 breast tumor universal normal controlPRO4339 colon tumor universal normal control PRO4326 lung tumoruniversal normal control PRO4326 colon tumor universal normal controlPRO6014 breast tumor universal normal control PRO3446 colon tumoruniversal normal control PRO3446 lung tumor universal normal controlPRO4322 lung tumor universal normal control PRO4322 rectal tumoruniversal normal control PRO4322 colon tumor matched normal coloncontrol PRO4381 breast tumor universal normal control PRO4381 lung tumoruniversal normal control PRO4381 colon tumor universal normal controlPRO4348 lung tumor universal normal control PRO4348 prostate tumoruniversal normal control PRO4371 breast tumor universal normal controlPRO3742 colon tumor universal normal control PRO3742 lung tumoruniversal normal control PRO5773 lung tumor universal normal controlPRO5773 colon tumor universal normal control PRO5773 prostate tumoruniversal normal control PRO5774 colon tumor universal normal controlPRO4343 colon tumor universal normal control PRO4325 lung tumoruniversal normal control PRO4347 lung tumor universal normal controlPRO4347 colon tumor universal normal control PRO4347 rectal tumoruniversal normal control PRO3743 colon tumor universal normal controlPRO3743 lung tumor universal normal control PRO3743 prostate tumoruniversal normal control PRO4426 colon tumor universal normal controlPRO4500 colon tumor universal normal control PRO4389 breast tumoruniversal normal control PRO4389 lung tumor universal normal controlPRO4337 colon tumor universal normal control PRO4337 breast tumoruniversal normal control PRO4337 lung tumor universal normal controlPRO4992 lung tumor universal normal control PRO5996 lung tumor universalnormal control PRO4345 lung tumor universal normal control PRO4345 colontumor universal normal control PRO5780 lung tumor universal normalcontrol PRO5780 breast tumor universal normal control PRO5992 lung tumoruniversal normal control PRO5992 colon tumor universal normal controlPRO5992 breast tumor universal normal control PRO4428 prostate tumoruniversal normal control PRO4994 lung tumor universal normal controlPRO5995 lung tumor universal normal control PRO5995 colon tumoruniversal normal control PRO6094 lung tumor universal normal controlPRO6094 colon tumor universal normal control PRO4317 lung tumoruniversal normal control PRO4317 colon tumor universal normal controlPRO4317 liver tumor universal normal control PRO4317 colon tumor matchednormal colon control PRO5997 colon tumor universal normal controlPRO5997 lung tumor universal normal control PRO5005 lung tumor universalnormal control PRO5005 colon tumor universal normal control PRO5004colon tumor universal normal control PRO6001 breast tumor universalnormal control PRO6013 colon tumor universal normal control PRO4502 lungtumor universal normal control PRO4502 colon tumor universal normalcontrol PRO6007 breast tumor universal normal control PRO6028 breasttumor universal normal control PRO6028 colon tumor universal normalcontrol PRO4327 prostate tumor universal normal control PRO4315 colontumor universal normal control PRO5993 lung tumor universal normalcontrol PRO5993 colon tumor universal normal control PRO4503 colon tumoruniversal normal control PRO4976 lung tumor universal normal controlPRO5798 lung tumor universal normal control PRO5798 colon tumoruniversal normal control PRO6242 colon tumor universal normal controlPRO6242 colon tumor matched normal colon control PRO6242 breast tumoruniversal normal control PRO6242 liver tumor universal normal controlPRO6242 rectal tumor universal normal control PRO6095 breast tumoruniversal normal control PRO6095 lung tumor universal normal controlPRO6093 colon tumor universal normal control PRO6093 breast tumoruniversal normal control PRO6093 lung tumor universal normal controlPRO6093 colon tumor matched normal colon control PRO6012 colon tumoruniversal normal control PRO6027 lung tumor universal normal controlPRO6027 colon tumor universal normal control PRO6027 rectal tumoruniversal normal control PRO6181 prostate tumor universal normal controlPRO6181 lung tumor universal normal control PRO6181 colon tumoruniversal normal control PRO6097 colon tumor universal normal controlPRO6097 lung tumor universal normal control PRO6090 lung tumor universalnormal control PRO7171 lung tumor universal normal control PRO7171 colontumor universal normal control PRO7171 breast tumor universal normalcontrol PRO6258 prostate tumor universal normal control PRO6258 breasttumor universal normal control PRO6258 cervical tumor universal normalcontrol PRO6258 liver tumor universal normal control PRO6258 colon tumoruniversal normal control PRO9820 prostate tumor universal normal controlPRO6243 lung tumor universal normal control PRO6182 lung tumor universalnormal control PRO6079 lung tumor universal normal control PRO6079 colontumor universal normal control PRO6079 breast tumor universal normalcontrol PRO6079 prostate tumor universal normal control PRO7434 lungtumor universal normal control PRO9865 colon tumor universal normalcontrol PRO9828 colon tumor universal normal control PRO196 colon tumoruniversal normal control PRO196 lung tumor universal normal controlPRO196 breast tumor universal normal control PRO197 colon tumoruniversal normal control PRO197 lung tumor universal normal controlPRO197 breast tumor universal normal control PRO195 colon tumoruniversal normal control PRO195 lung tumor universal normal controlPRO195 breast tumor universal normal control PRO187 lung tumor universalnormal control PRO187 liver tumor universal normal control PRO182 colontumor universal normal control PRO182 lung tumor universal normalcontrol PRO182 breast tumor universal normal control PRO188 rectal tumoruniversal normal control PRO183 colon tumor universal normal controlPRO183 lung tumor universal normal control PRO183 breast tumor universalnormal control PRO183 rectal tumor universal normal control PRO184 lungtumor universal normal control PRO184 breast tumor universal normalcontrol PRO185 lung tumor universal normal control PRO200 colon tumoruniversal normal control PRO200 lung tumor universal normal controlPRO200 breast tumor universal normal control PRO200 rectal tumoruniversal normal control PRO202 colon tumor universal normal controlPRO202 lung tumor universal normal control PRO202 breast tumor universalnormal control PRO202 rectal tumor universal normal control PRO202 livertumor universal normal control PRO214 colon tumor universal normalcontrol PRO214 lung tumor universal normal control PRO215 colon tumoruniversal normal control PRO215 lung tumor universal normal controlPRO215 breast tumor universal normal control PRO219 colon tumoruniversal normal control PRO219 lung tumor universal normal controlPRO219 breast tumor universal normal control PRO219 liver tumoruniversal normal control PRO211 lung tumor universal normal controlPRO211 breast tumor universal normal control PRO220 colon tumoruniversal normal control PRO220 lung tumor universal normal controlPRO220 breast tumor universal normal control PRO366 colon tumoruniversal normal control PRO366 lung tumor universal normal controlPRO366 breast tumor universal normal control PRO216 lung tumor universalnormal control PRO221 colon tumor universal normal control PRO221 lungtumor universal normal control PRO221 breast tumor universal normalcontrol PRO228 lung tumor universal normal control PRO228 breast tumoruniversal normal control PRO217 lung tumor universal normal controlPRO217 breast tumor universal normal control PRO222 colon tumoruniversal normal control PRO222 lung tumor universal normal controlPRO222 breast tumor universal normal control PRO224 colon tumoruniversal normal control PRO224 lung tumor universal normal controlPRO224 breast tumor universal normal control PRO224 prostate tumoruniversal normal control PRO224 rectal tumor universal normal controlPRO230 colon tumor universal normal control PRO230 lung tumor universalnormal control PRO230 breast tumor universal normal control PRO230prostate tumor universal normal control PRO198 colon tumor universalnormal control PRO198 lung tumor universal normal control PRO198 breasttumor universal normal control PRO198 liver tumor universal normalcontrol PRO226 lung tumor universal normal control PRO226 breast tumoruniversal normal control PRO261 lung tumor universal normal controlPRO242 colon tumor universal normal control PRO242 lung tumor universalnormal control PRO242 breast tumor universal normal control PRO227 colontumor universal normal control PRO227 lung tumor universal normalcontrol PRO237 colon tumor universal normal control PRO237 lung tumoruniversal normal control PRO237 breast tumor universal normal controlPRO237 prostate tumor universal normal control PRO241 colon tumoruniversal normal control PRO241 lung tumor universal normal controlPRO241 breast tumor universal normal control PRO231 colon tumoruniversal normal control PRO231 lung tumor universal normal controlPRO231 breast tumor universal normal control PRO231 rectal tumoruniversal normal control PRO235 colon tumor universal normal controlPRO235 lung tumor universal normal control PRO235 breast tumor universalnormal control PRO235 liver tumor universal normal control PRO323 lungtumor universal normal control PRO323 breast tumor universal normalcontrol PRO323 rectal tumor universal normal control PRO245 colon tumoruniversal normal control PRO245 lung tumor universal normal controlPRO245 breast tumor universal normal control PRO245 cervical tumoruniversal normal control PRO245 liver tumor universal normal controlPRO246 colon tumor universal normal control PRO246 lung tumor universalnormal control PRO246 breast tumor universal normal control PRO288 lungtumor universal normal control PRO288 breast tumor universal normalcontrol PRO248 lung tumor universal normal control PRO248 rectal tumoruniversal normal control PRO257 colon tumor universal normal controlPRO257 lung tumor universal normal control PRO257 prostate tumoruniversal normal control PRO172 colon tumor universal normal controlPRO172 lung tumor universal normal control PRO172 breast tumor universalnormal control PRO258 colon tumor universal normal control PRO258 lungtumor universal normal control PRO258 breast tumor universal normalcontrol PRO265 lung tumor universal normal control PRO265 breast tumoruniversal normal control PRO265 rectal tumor universal normal controlPRO326 colon tumor universal normal control PRO326 lung tumor universalnormal control PRO326 breast tumor universal normal control PRO326 livertumor universal normal control PRO266 colon tumor universal normalcontrol PRO266 lung tumor universal normal control PRO266 breast tumoruniversal normal control PRO269 lung tumor universal normal controlPRO269 rectal tumor universal normal control PRO285 colon tumoruniversal normal control PRO285 lung tumor universal normal controlPRO285 breast tumor universal normal control PRO328 colon tumoruniversal normal control PRO328 lung tumor universal normal controlPRO328 breast tumor universal normal control PRO344 breast tumoruniversal normal control PRO272 lung tumor universal normal controlPRO301 colon tumor universal normal control PRO301 lung tumor universalnormal control PRO301 breast tumor universal normal control PRO331 colontumor universal normal control PRO331 lung tumor universal normalcontrol PRO331 breast tumor universal normal control PRO332 colon tumoruniversal normal control PRO332 lung tumor universal normal controlPRO332 breast tumor universal normal control PRO353 colon tumoruniversal normal control PRO353 lung tumor universal normal controlPRO353 breast tumor universal normal control PRO310 colon tumoruniversal normal control PRO310 lung tumor universal normal controlPRO310 breast tumor universal normal control PRO310 rectal tumoruniversal normal control PRO337 colon tumor universal normal controlPRO337 lung tumor universal normal control PRO337 breast tumor universalnormal control PRO346 lung tumor universal normal control PRO350 lungtumor universal normal control PRO350 breast tumor universal normalcontrol PRO526 colon tumor universal normal control PRO526 lung tumoruniversal normal control PRO526 breast tumor universal normal controlPRO381 colon tumor universal nonnal control PRO381 lung tumor universalnormal control PRO381 breast tumor universal normal control PRO381prostate tumor universal normal control PRO846 colon tumor universalnormal control PRO846 lung tumor universal normal control PRO363 colontumor universal normal control PRO363 lung tumor universal normalcontrol PRO365 lung tumor universal normal control PRO365 breast tumoruniversal normal control PRO1310 breast tumor universal normal controlPRO731 colon tumor universal normal control PRO731 lung tumor universalnormal control PRO731 breast tumor universal normal control PRO322 colontumor universal normal control PRO322 lung tumor universal normalcontrol PRO322 breast tumor universal normal control PRO322 rectal tumoruniversal normal control PRO322 liver tumor universal normal controlPRO536 lung tumor universal normal control PRO536 breast tumor universalnormal control PRO536 liver tumor universal normal control PRO719 colontumor universal normal control PRO719 lung tumor universal normalcontrol PRO719 breast tumor universal normal control PRO619 colon tumoruniversal normal control PRO619 lung tumor universal normal controlPRO619 breast tumor universal normal control PRO771 colon tumoruniversal normal control PRO771 lung tumor universal normal controlPRO771 breast tumor universal normal control PRO1083 colon tumoruniversal normal control PRO1083 lung tumor universal normal controlPRO1083 breast tumor universal normal control PRO1083 prostate tumoruniversal normal control PRO862 colon tumor universal normal controlPRO862 lung tumor universal normal control PRO862 breast tumor universalnormal control PRO733 colon tumor universal normal control PRO733 lungtumor universal normal control PRO733 breast tumor universal normalcontrol PRO733 liver tumor universal normal control PRO1188 lung tumoruniversal normal control PRO1188 breast tumor universal normal controlPRO1188 rectal tumor universal normal control PRO770 lung tumoruniversal normal control PRO770 breast tumor universal normal controlPRO1080 colon tumor universal normal control PRO1080 lung tumoruniversal normal control PRO1080 breast tumor universal normal controlPRO1017 colon tumor universal normal control PRO1017 lung tumoruniversal normal control PRO1017 breast tumor universal normal controlPRO1016 colon tumor universal normal control PRO1016 lung tumoruniversal normal control PRO1016 breast tumor universal normal controlPRO1016 rectal tumor universal normal control PRO792 lung tumoruniversal normal control PRO938 colon tumor universal normal controlPRO938 lung tumor universal normal control PRO938 breast tumor universalnormal control PRO1012 colon tumor universal normal control PRO1012 lungtumor universal normal control PRO1012 rectal tumor universal normalcontrol PRO1012 liver tumor universal normal control PRO1008 lung tumoruniversal normal control PRO1075 colon tumor universal normal controlPRO1075 lung tumor universal normal control PRO1007 colon tumoruniversal normal control PRO1007 lung tumor universal normal controlPRO1007 breast tumor universal normal control PRO1007 rectal tumoruniversal normal control PRO1056 colon tumor universal normal controlPRO1056 lung tumor universal normal control PRO1056 breast tumoruniversal normal control PRO791 colon tumor universal normal controlPRO791 lung tumor universal normal control PRO791 breast tumor universalnormal control PRO791 rectal tumor universal normal control PRO1111colon tumor universal normal control PRO1111 lung tumor universal normalcontrol PRO1111 breast tumor universal normal control PRO812 lung tumoruniversal normal control PRO812 breast tumor universal normal controlPRO812 rectal tumor universal normal control PRO1066 lung tumoruniversal normal control PRO1185 colon tumor universal normal controlPRO1185 lung tumor universal normal control PRO1185 breast tumoruniversal normal control PRO1031 lung tumor universal normal controlPRO1360 lung tumor universal normal control PRO1360 breast tumoruniversal normal control PRO1309 lung tumor universal normal controlPRO1309 breast tumor universal normal control PRO1107 lung tumoruniversal normal control PRO1107 breast tumor universal normal controlPRO836 colon tumor universal normal control PRO836 lung tumor universalnormal control PRO1132 lung tumor universal normal control PRO1132breast tumor universal normal control PRO1131 colon tumor universalnormal control PRO1131 lung tumor universal normal control PRO1131breast tumor universal normal control PRO1131 liver tumor universalnormal control PRO1130 colon tumor universal normal control PRO1130 lungtumor universal normal control PRO1130 breast tumor universal normalcontrol PRO844 colon tumor universal normal control PRO844 lung tumoruniversal normal control PRO844 breast tumor universal normal controlPRO844 rectal tumor universal normal control PRO1154 colon tumoruniversal normal control PRO1154 lung tumor universal normal controlPRO1154 rectal tumor universal normal control PRO1154 liver tumoruniversal normal control PRO1181 lung tumor universal normal controlPRO1181 breast tumor universal normal control PRO1126 colon tumoruniversal normal control PRO1126 lung tumor universal normal controlPRO1126 breast tumor universal normal control PRO1126 adrenal tumoruniversal normal control PRO1186 colon tumor universal normal controlPRO1186 lung tumor universal normal control PRO1186 breast tumoruniversal normal control PRO1186 liver tumor universal normal controlPRO1198 colon tumor universal normal control PRO1198 lung tumoruniversal normal control PRO1159 lung tumor universal normal controlPRO1159 breast tumor universal normal control PRO1159 liver tumoruniversal normal control PRO1265 colon tumor universal normal controlPRO1265 breast tumor universal normal control PRO1250 colon tumoruniversal normal control PRO1250 lung tumor universal normal controlPRO1250 breast tumor universal normal control PRO1475 colon tumoruniversal normal control PRO1475 breast tumor universal normal controlPRO1312 colon tumor universal normal control PRO1312 lung tumoruniversal normal control PRO1312 breast tumor universal normal controlPRO1308 colon tumor universal normal control PRO1308 lung tumoruniversal normal control PRO1308 liver tumor universal normal controlPRO1326 colon tumor universal normal control PRO1325 lung tumoruniversal normal control PRO1326 breast tumor universal normal controlPRO1192 colon tumor universal normal control PRO1192 lung tumoruniversal normal control PRO1192 breast tumor universal normal controlPRO1246 colon tumor universal normal control PRO1246 lung tumoruniversal normal control PRO1246 breast tumor universal normal controlPRO1246 prostate tumor universal normal control PRO1356 colon tumoruniversal normal control PRO1356 lung tumor universal normal controlPRO1356 breast tumor universal normal control PRO1275 lung tumoruniversal normal control PRO1275 breast tumor universal normal controlPRO1274 lung tumor universal normal control PRO1358 colon tumoruniversal normal control PRO1358 lung tumor universal normal controlPRO1358 prostate tumor universal normal control PRO1286 colon tumoruniversal normal control PRO1286 lung tumor universal normal controlPRO1286 prostate tumor universal normal control PRO1286 rectal tumoruniversal normal control PRO1294 colon tumor universal normal controlPRO1294 lung tumor universal normal control PRO1294 breast tumoruniversal normal control PRO1294 rectal tumor universal normal controlPRO1273 lung tumor universal normal control PRO1273 rectal tumoruniversal normal control PRO1279 colon tumor universal normal controlPRO1279 lung tumor universal normal control PRO1195 lung tumor universalnormal control PRO1195 breast tumor universal normal control PRO1271lung tumor universal normal control PRO1271 breast tumor universalnormal control PRO1271 liver tumor universal normal control PRO1338colon tumor universal normal control PRO1338 lung tumor universal normalcontrol PRO1338 breast tumor universal normal control PRO1343 colontumor universal normal control PRO1343 lung tumor universal normalcontrol PRO1343 breast tumor universal normal control PRO1343 rectaltumor universal normal control PRO1434 lung tumor universal normalcontrol PRO1418 lung tumor universal normal control PRO1418 liver tmnoruniversal normal control PRO1387 colon tumor universal normal controlPRO1387 lung tumor universal normal control PRO1387 prostate tumoruniversal normal control PRO1387 rectal tumor universal normal controlPRO1384 colon tumor universal normal control PRO1384 lung tumoruniversal normal control PRO1565 colon tumor universal normal controlPRO1565 lung tumor universal normal control PRO1565 prostate tumoruniversal normal control PRO1474 colon tumor universal normal controlPRO1474 lung tumor universal normal control PRO1474 breast tumoruniversal normal control PRO1474 rectal tumor universal normal controlPRO1917 colon tumor universal normal control PRO1917 lung tumoruniversal normal control PRO1917 breast tumor universal normal controlPRO1787 colon tumor universal normal control PRO1787 lung tumoruniversal normal control PRO1787 breast tumor universal normal controlPRO1556 lung tumor universal normal control PRO1556 breast tumoruniversal normal control PRO1561 colon tumor universal normal controlPRO1561 lung tumor universal normal control PRO1561 rectal tumoruniversal normal control PRO1693 colon tumor universal normal controlPRO1693 lung tumor universal normal control PRO1693 breast tumoruniversal normal control PRO1868 lung tumor universal normal controlPRO1868 breast tumor universal normal control PRO1890 colon tumoruniversal normal control PRO1890 lung tumor universal normal controlPRO1890 breast tumor universal normal control PRO1890 prostate tumoruniversal normal control PRO1887 colon tumor universal normal controlPRO1887 breast tumor universal normal control PRO4353 lung tumoruniversal normal control PRO4353 breast tumor universal normal controlPRO1801 colon tumor universal normal control PRO1801 lung tumoruniversal normal control PRO4357 lung tumor universal normal controlPRO4357 breast tumor universal normal control PRO4302 colon tumoruniversal normal control PRO4302 lung tumor universal normal controlPRO4302 breast tumor universal normal control PRO4302 prostate tumoruniversal normal control PRO5990 colon tumor universal normal controlPRO5990 lung tumor universal normal control PRO5990 breast tumoruniversal normal control

Example 31 Identification of Receptor/Ligand Interactions

In this assay, various PRO polypeptides are tested for ability to bindto a panel of potential receptor or ligand molecules for the purpose ofidentifying receptor/ligand interactions. The identification of a ligandfor a known receptor, a receptor for a known ligand or a novelreceptor/ligand pair is useful for a variety of indications including,for example, targeting bioactive molecules (linked to the ligand orreceptor) to a cell known to express the receptor or ligand, use of thereceptor or ligand as a reagent to detect the presence of the ligand orreceptor in a composition suspected of containing the same, wherein thecomposition may comprise cells suspected of expressing the ligand orreceptor, modulating the growth of or another biological orimmunological activity of a cell known to express or respond to thereceptor or ligand, modulating the immune response of cells or towardcells that express the receptor or ligand, allowing the preparaion ofagonists, antagonists and/or antibodies directed against the receptor orligand which will modulate the growth of or a biological orimmunological activity of a cell expressing the receptor or ligand, andvarious other indications which will be readily apparent to theordinarily skilled artisan.

The assay is performed as follows. A PRO polypeptide of the presentinvention suspected of being a ligand for a receptor is expressed as afusion protein containing the Fc domain of human IgG (an immunoadhesin).Receptor-ligand binding is detected by allowing interaction of theimmunoadhesin polypeptide with cells (e.g. Cos cells) expressingcandidate PRO polypeptide receptors and visualization of boundimmunoadhesin with fluorescent reagents directed toward the Fc fusiondomain and examination by microscope. Cells expressing candidatereceptors are produced by transient transfection, in parallel, ofdefined subsets of a library of cDNA expression vectors encoding PROpolypeptides that may function as receptor molecules. Cells are thenincubated for 1 hour in the presence of the PRO polypeptideimmunoadhesin being tested for possible receptor binding. The cells arethen washed and fixed with paraformaldehyde. The cells are thenincubated with fluorescent conjugated antibody directed against the Fcportion of the PRO polypeptide immunoadhesin (e.g. FITC conjugated goatanti-human-Fc antibody). The cells are then washed again and examined bymicroscope. A positive interaction is judged by the presence offluorescent labeling of cells transfected with cDNA encoding aparticular PRO polypeptide receptor or pool of receptors and an absenceof similar fluorescent labeling of similarly prepared cells that havebeen transfected with other cDNA or pools of cDNA. If a defined pool ofcDNA expression vectors is judged to be positive for interaction with aPRO polypeptide immunoadhesin, the individual cDNA species that comprisethe pool are tested individually (the pool is “broken down”) todetermine the specific cDNA that encodes a receptor able to interactwith the PRO polypeptide immunoadhesin.

In another embodiment of this assay, an epitope-tagged potential ligandPRO polypeptide (e.g. 8 histidine “His” tag) is allowed to interact witha panel of potential receptor PRO polypeptide molecules that have beenexpressed as fusions with the Fc domain of human IgG (immunoadhesins).Following a 1 hour co-incubation with the epitope tagged PROpolypeptide, the candidate receptors are each immunoprecipitated withprotein A beads and the beads are washed. Potential ligand interactionis determined by western blot analysis of the immunoprecipitatedcomplexes with antibody directed towards the epitope tag. An interactionis judged to occur if a band of the anticipated molecular weight of theepitope tagged protein is observed in the western blot analysis with acandidate receptor, but is not observed to occur with the other membersof the panel of potential receptors.

Using these assays, the following receptor/ligand interactions have beenherein identified:

-   (1) PRO1801 binds to PRO1114 and PRO4978.-   (2) PRO100 binds to PRO1114.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated antibody that specifically binds to the polypeptide ofSEQ ID NO:426.
 2. The antibody of claim 1 which is a monoclonalantibody.
 3. The antibody of claim 1 which is a humanized antibody. 4.The antigen-binding fragment of the antibody of claim
 1. 5. The antibodyof claim 1 which is labeled.